TWI297731B - Bleb preparation from neisseria meningitidis strain - Google Patents

Description

1297731 Intellectual Property Office of the Ministry of Economic Affairs, Printed by the Consumer Cooperatives A7 B7 V. INSTRUCTIONS (1) Field of the Invention The present invention relates to the field of gram-negative bacterial vaccine compositions, their manufacture, and their use in medicine. More particularly, it relates to the field of novel outer membrane vesicles (or large vesicles) vaccines, and to beneficial methods that make them more effective and safer. BACKGROUND OF THE INVENTION Gramella is separated from the external medium by a two-layer continuous membrane structure. These structures are called cytoplasmic and outer membranes (0M) and differ in structure and function. The outer membrane plays an important role in the interaction of pathogenic bacteria with their individual hosts. Therefore, surface-exposed bacterial molecules are an important target for host immune responses, making the outer membrane component an attractive candidate for providing vaccines, diagnostics, and therapeutic agents. Whole cell bacterial vaccines (dead or attenuated) have the advantage of providing multiple antigens in their natural microbial environment. The difficulties encountered in this pathway are side effects caused by bacterial components such as endotoxin and peptide polysaccharide fragments. On the other hand, the bacterial-free subunit vaccine containing the purified component from the outer membrane provides only limited protection and may not have an antigen suitable for the host immune system. Proteins 'phospholipids and lipopolysaccharides are the three major components found in the outer membrane of all Gram-negative bacteria. These molecules are asymmetrically distributed · membrane phospholipids (mostly in internal folds), lipooligosaccharides (not included in external folds) and proteins (internal and exogenous sclerotin proteins, intact or multiplexed membrane proteins) . For the bacterial pathogens of Xuxi, which impact human health, it has been shown that lipopolysaccharide and outer membrane proteins are immunogenic and are easy to protect against considerable diseases by immunization. _____ - 4 _ G Zhang scale applies China i豕 standard (CNS) A4 rule _ (10) χ tear public love) h---1----r--------- 11--r 111 ^i I----- (Please read the note on the back? Please fill out this page again) 1297731 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7__ V. Invention description (2) Gram-negative bacteria Ο Yes Dynamic, and depending on environmental conditions, dramatic pathology can be performed. In these characterizations, the formation of outer membrane vesicles or "big vesicles" has been studied and demonstrated in many Gram-negative bacteria (Zhou, L et al. 1998, FEMS Microbiol. Lett. 163: 223-228). Among them, the bacterial pathogens are reported to be incompletely produced to produce large blisters, including: Bordetella pertussis, Borrelia burgdorferi, Brucella melitensis ), Brucella ovis, Chlamydia psittaci, Chlamydia trachomatis, Esherichia coli, Haemophilus influenzae, Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa and Yersinia enterocolitica. Although the biochemical mechanisms responsible for the production of blister blister are not fully understood, these outer membrane vesicles have been adequately studied because they represent a powerful methodology to isolate outer membrane protein preparations in their native configuration. In this environment, the development of a vaccine against Neisserella, Moraxella catarrhalis, Haemophilus influenzae, Pseudomonas aeruginosa and Chlamydia using an outer membrane preparation is of particular interest. Furthermore, the outer membrane large blister mixed with multiple proteins and non-protein antigens appears to provide a more extended protection against antagonistic species. Compared to other bacterial vaccines that are more widely used (whole cell bacteria and purified subunit vaccines), the inventors have shown that the outer membrane large blister vaccine (if changed in some respects) may represent an ideal compromise the way. -5- _This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm) ---^---- - - ---------- I- ^ · (Read first 1233 31 A7 V. Inventive Note (3) Wide application of bacterial subunit vaccines ^ Due to the study of bacterial surface egg filling, it has been found to be very useful in vaccine application. Pertussis protein of Bordetella. These proteins and fines are not large and can be purified from culture supernatants or easily extracted from bacterial cells. However, structurally intact membrane proteins have been shown. It is also a protective antigen. Examples are Neisseria meningitidis serum B group ^ heart A; Haemophilus influenzae D i 5 ; Moraxella catarrhalis (M. OMP CD; Pseudomonas aeruginosa) Mp F. However, this protein has rare and specific structural features, especially multiple amphiphilic layers, making their direct use of purified (recombinant) subunit vaccines more complicated. When a reasonable level of protection is supplied, multiple component vaccines are needed (for bacteria) For surface proteins and intact membrane proteins). For example, in the case of the B. pertussis subunit vaccine, the multi-reconstituted vaccine is superior to the single or two-component product. In order to incorporate the intact outer membrane protein into this sub- In the unit product, the product must have a natural (or nearly natural) configuration of the protein folded to have a useful immune effect. The use of a membranous sac or large blisters may be a wonderful solution to the following problems, which is about to be completed. The protective membrane protein is included in the subunit vaccine, while still ensuring that it has the correct folding pattern. The meningitis group B (menB) can discharge a sufficient amount of large blister of the outer membrane to make it Industrial-scale manufacturing. The multi-component outer membrane protein vaccine obtained from the naturally occurring menB strain was found to be effective in protecting children in their teens from menb disease and has been greened in Latin America. Another method of sac is to remove the sputum from the bacterial cells (EP 丨 1243) — — — — — —• 丨·. (Please read the notes on the back and fill in This page) tT---------. Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed -6-1297731 A7 _______B7_____ V. Invention Description (4) Take the procedure. Examples of bacteria that can be made into a large blister vaccine are as follows · Neisseria meningitidis Neisseria meningitidis is a Gram-negative bacterium that is often isolated from the human upper respiratory tract. It often causes invasive bacterial diseases such as bacteremia and meningitis. Meningococcal disease Incidence shows geographic seasonality and annual differences (Schwartz, B., Moore, P. S. Broome, C. V.; Clin·Microbiol Rev. 2 (Supplement), S18-S24, 1989). Most diseases in temperate countries are caused by serogroup B strains, which vary from one to 1-10/100,000 per year in the total population and can sometimes be as high as 値 (Kaczmarski, EB (1997), Commun· Dis· Rep· Rev. 7: R55-9, 1995; Scholten, R. J. P. M., Bijlmer, HA, Poolman, JT et al. Clin· Infect· Dis· 16: 237-246, 1993; Cruz, C ·, Pavez, G., Aguilar, E., et al. Epidemiol· Infect. 105: 119-126, 1990). In the two high-risk groups with age-specific incidence, infants and children in their teens can reach higher levels. The predominance of serogroup A meningococcal disease occurs mostly in Central Africa, sometimes as high as 1000/100,000/year (Schwartz, B., Moore, PS, Broome, CV Clin. Microbiol. Rev. 2 ( Supplement), SI8-S24, 1989). Overall, almost all cases of meningococcal disease are caused by A, B, C, W-135 and Y meningococcal serotypes. A tetravalent A, C, W-135, Y capsular vinegar vaccine can be applied (Armand, J., Arminjon, F., Mynard, M. C., Lafaix, C., J. Biol. Stand. 10 : 335-339, 1982). Polysaccharide vaccines are currently improved by chemical conjugation to carrier protein methods. This paper scale applies to China National Standard (CNS) A4 specifications (210 X 297 mm). i 4i-------------- (Please Read the following notes on the back and fill out this page) tr---------MW, Ministry of Economic Affairs, Intellectual Property Bureau, Staff and Consumers Cooperative, Ministry of Printing, Ministry of Economy, Intellectual Property Office, Staff and Consumers Cooperative, Printed 1297731 A7 ___B7___ V. Invention Description (5) (Lieberman, J. M., Chiu, S. S., Wong, V. K., et al., JAMA 275: l499-l5〇3, 1996) Serotype vaccines are not available because b laughs Membrane polysaccharides are non-immunogenic, most likely because they share structural similarities with host components (Wyle, FA, Artenstein, M. S., Brandt, M. L. et al. J. Infect· Dis· 126: 514-522, 1972; Finne, J. M., Leinonen, M., MSkeH, Ρ·M·Lancet ii: 355-357, 1983) o Efforts over the years have focused on the development of meningococcal Membrane-based vaccines (de Moraes, J. C., Perkins, B., Camargo, MC et al. Lancet 340: 1074-1078, 1992; Bjune, G., Hoiby, E. A. Gronnesby, J. K. et al. 33 8: 1093-1096, 1991). This vaccine has proven to be effective in older children (> 4 years old) and adolescent youth, from 57% to 85%. Most of these efficacy trials were performed with a vaccine of OMV (outer capsular sac, derived from large blister-depleted LPS) derived from the wild-type N. meningitidis B strain. Many bacterial outer membrane components are present in these vaccines, such as PorA, PorB, Rmp, Opc, Opa, FrpB, and the contribution of these components to the observed protective effects needs to be further defined. Other bacterial outer membrane components have been identified (using animal and human antibodies) for possible association with protective immunity, such as TbpB, NspA (Martin, D., Cadieux, N., Hamel, J., Brodeux, B. R., J. Exp. Med. 185: 1 173-1 183, 1997; Lissolo, L., Maitre-Wilmotte, C., Dumas, p. et al., Inf· Immun· 63: 884-890 , 1995). The mechanism of protecting immunity involves the bacteriostatic activity mediated by antibodies and the regulation of phagocytosis. The frequency of Neisseria meningitidis infection, dramatic since the past several -8 - This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) !! φ· — — 订----- ---- (Please read the note on the back and then fill out this page) 1297731 A7, invention description (6) = due to the emergence of multiple antibiotic resistant strains and increased exposure due to social activities (such as swimming pool or theater). Separation of the meninges = (2) The ball is no longer unusual, and it has several properties for some or all of the standard antibiotics. This phenomenon has generated the medical needs of unmet, and has new antibacterial agents, vaccines, drug screening methods, and diagnostic tests for this organism t. 0. Moraxella (also known as B. blancheri (10) Qtarrhalis is a Gram-negative bacterium, often isolated from human upper respiratory f. It is responsible for many pathologies, the main diseases being otitis in infants and children and older pneumonia. It is also associated with sinusitis, infections in hospitals, and less invasive diseases. Most of the adults tested were found to have bacteriostatic antibodies (Chapman, AJ et al. (1985) J. lnfect· Dis. 151:878). Moraxella has a diversity in the ability to resist bacterial bacteriostatic activity: in general, isolates obtained from diseased individuals are more resistant than those from simple colonies (H〇1·C et spit ( 1993) Lancet 341:1281, Jordan, KL et al. (1990) Am. J. Med. 88 (suppl·5A): 28S). Therefore, serum resistance can be considered as a virulence factor of bacteria. Conditioning activity was observed in children's serum recovered from otitis media. The antigens that can be targeted for these different immune responses in humans have not been identified, with the exception of OMP B1, an 84 kDa protein whose expression is regulated by iron and can be confirmed by the serum of patients with pneumonia (Sethi, s. et Al. (1995) Infect. Immun. 63:1516), and UspAl and UspA2 (Chen D. et al. (1999), Infect. Immun. 67: 1310). -9 - This paper size is applicable to China National Standard (CNS) A4 specification (210 x 297 mm) Γ ---------Φ! Order·------ (Please read the notes on the back first) Fill in this page again) Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1297731 A7 B7 V. Invention description (7) Biochemical methods have been used to identify some other membrane proteins present on the surface of Moraxella catarrhalis, to study its breeding Possible roles in protective immunity (for a general overview, see Murphy, TF (1996) Microbiol Rev. 60:267) In the mouse pneumonia model, some of the antibodies produced (UspA, CopB) are involved in lung infection. There is a faster clearance rate. Another polypeptide (OMP CD) is highly retained in Moraxella catarrhalis and exhibits homogeneity with Pseudomonas aeruginosa, which has been shown to be effective against antagonism in animal models. Moraxella catarrhalis produces an outer sac (large blister). These large blisters have been isolated or extracted using different methods (Murphy T. F., Loeb M. R. 1989. Microb. Pathog. 6: 159-174; Unhanand M., Maciver, I., Ramillo 0., Arencibia-Mireles 0·, Argyle J. C., McCracken GH Jr., Hansen E. J. 1992. J. Infect· Dis· 165: 644-650). The protective power of this large leeches has been tested in the murine Moxies for the lung clearance rate of Moraxella catarrhalis. It has been shown that active immunization with a large blister vaccine or passive transfer with anti-big blister antibodies can have significant protective effects in the model (Maciver I., Unhanand M., McCracken GH Jr., Hansen, E. J) 1993· J· Infect· Dis. 168: 469-472). Haemophilus influenzae Haemophilus influenzae is an incompetent gram-negative bacterium. People are the only natural hosts. Haemophilus influenzae isolates are usually classified by their polysaccharide membranes. A total of six different film types from a' to π have been identified. Aliquots that are unable to agglutinate with the antiserum antagonized by one of the six serotypes are classified in a group that cannot be typed and do not exhibit a sandwich. Hemophilus influenzae type b (Hib) is clearly different from other types because it is 10--paper scale applicable to China National Standard (CNS) A4 specification (210 X 297 mm) Γ I1III — — — — — — — II (Please read the note on the back and then fill out this page) Order -------------MW, Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperative, Printed 12,773,371 Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperatives System A7 B7 V. Description of the invention (8) The main cause of bacterial meningitis and systemic diseases. The incompetent strain of H. influenzae (NTHi) is isolated only from the blood of patients with systemic diseases. NTHi is a common cause of pneumonia, chronic bronchitis, sinusitis and otitis media. It was identified by pure line analysis that the NTHi strain had large variability, while the Hib strain was homogeneous as a whole. Various proteins of H. influenzae have been shown to be involved in the pathogenesis principle or to provide protection once immunized into an animal. The adhesion of NTHi to human nasopharyngeal epithelial cells has been reported (Read RC. et al. 1991 J Infect· Dis· 163:549). In addition to fine hairs and cilia (Brinton CC. et al. 1989. Pediatr. Infect. Dis. J. 8:S54; Kar S. et al. 1990. Infect. Immun. 58:903; Gildorf JR. et al. 1992. Infect. Immun. 60:374; St. Geme JW et al. 1991. Infect. Immun. 59:3366; St. Geme JW et al. 1993. Infect. Immun. 61: 2233), many adhesins in NTHi It has been known. Two high-molecular-weight proteins with surface exposure are shown here. HMW1 and HMW2 can upregulate the adhesion of NTHi to epithelial bacteria (St. Geme JW. et al. 1993. Proc. Natl. Acad. Sci. USA 90:2875 ). Another family of high molecular weight proteins has been identified in the NTHi strain, which lacks proteins belonging to the HMW1/HMW2 family. NTHi 115-kDa Hia protein (Barenkamp SJ·, St Geme S. W. 1996. Mol·MicrobioH) is highly similar to Hsf adhes expressed by Haemophilus influenzae type b strain (St. Geme JW. etal · 1996· J. Bact. 178:6281). Another protein, Hap protein and IgA 1 serine protease, is similar and is shown to be involved in adhesion and bacterial entry. (St. Geme JW. et al. 1994. Mol·Microbiol· 14:217). -11 - This paper size is applicable to China National Standard (CNS) A4 specification (210 x 297 mm) -I J----------- II ! (Please read the note on the back and fill out this page) tr ---------3W, Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1297731 A7 _B7__ V. Description of invention (9) The five major outer membrane proteins (OMP) have been identified and numbered. Previous studies using H. influenzae type B strains have shown that antibodies specific for P1 and P2 OMPs protect infant rats from subsequent elaboration (Loeb MR. et al. 1987. Infect. Immun. 55:2612) Musson RS. Jr. et al. 1983. J. Clin·Invest· 72:677) It has been found that P 2 can produce bacteriostatic and opsonic antibodies that directly antagonize the ring structure that is present on the surface of this intact OMP. Variable region (Haase EM. et al. 1994 Infect. Immun. 62:3712; Troelstra A. et al. 1994 Infect. Immun. 62:779). Lipoprotein P 4 can also produce bacterial antibodies (Green BA. et al. 1991 · Infect· Immun. 59:3 191). OMP P6 is a retentive peptide-polysaccharide-linked lipoprotein that occupies 1-5% of the outer membrane (Nelson MB. et al. 1991. Infect. Immun 59: 2658). A lipoprotein of approximately the same molecular weight was identified as PCP (P6 interacting protein) (Deich RM. et al. 1990. Infect. Immun. 58:3388). The retained lipoproteins P4, P6 and PCP mixtures did not show protection in the chinchilla otitis media pattern (Green BA. et al. 1993· Infect, immun· 61 · 1950). P 6 alone appears to have protective effects in the chinchilla mode (Demaria TF· et al· 1996· Infect· Immun. 64:5187) ° Fine hair protein (Miyamoto N·, Bakaletz, LO. 1996· Microb· ?&; (^.21:343) is also described as homogenous to 〇]^??5, which itself has sequence homogeneity with the complete E. coli OmpA (Miyamoto N., Bakaletz, LO. 1996. Microb. Pathog. 21:343; Munson RS. Jr. et al. 1993. Infect· Immun· 61:1017). NTHi appears to adhere to the mucosa via fine hair. Compared with the observations using gonorrhea and meningococcus, NTHi is at -12- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) !! l-----------i — order---------- 3W (please first Read the notes on the back and fill out this page.) 1297731 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed A7 B7 V. Invention Description (ίο ) Growth under iron-limited conditions, showing double human iron composed of TbpA and TbpB Transfer protein receptor. Anti-TbpB antibody protects young rats (Loosmore SM. et al. 1996. Mol·Microbiol. 19:575). Hemoglobin/ The globulin receptor is also mentioned in NTHi (Maciver I. et al. 1996. Infect. Immun. 64:3703). The hemorrhagic matrix is also identified: the receptor for hemagglutinin (Haem: Hemopexin) (Cope LD. et Al·1994· Mol·Microbiol. 13:868). The lactate receptor is also between NTHi but not yet identified (Schryvers AB· et al· 1989· J. Med. Microbiol. 29:121) 〇 in NHTi yet A protein similar to the N. faecalis FrpB-protein is described. 80kDa OMP, D15 surface antigen, provides protection against NTHi in a mouse challenge mode (Flack FS· et al· 1995· Gene 156:97) 〇42kDa Membrane lipoprotein, LPD, is retained in H. influenzae and can induce the production of bacteriostatic antibodies (Akkoyunlu M. et al. 1996. Infect. Immun. 64: 4586). Less 98kDa OMP (Kimura A. et al. 1985. Infect. Immun. 47:253) was found to be a protective antigen, and this OMP is most likely one of the OMPs induced by Fe-restricted or later identified. High molecular weight adhesin. Haemophilus influenzae produces IgAl-protease activity (Mulks MH., Shoberg RJ. 1994. Meth. Enzymol. 235: 543). NTHi's IgAl-proteinase has a high degree of antigenic variability (Lomholt H., van Alphen L., Kilian, M. 1993. Infect. Immun 61:4575). Another OMP of NHTi, OMP26, is a 26-kDa protein that has been shown to enhance lung clearance in rat mode (1^(1, 〖.1^., €:1^08, 八·W) 1998· Infect· Immun·66:2272). It also shows that NTHi HtrA protein is a protective antigen. Indeed, the protein can protect the chinchilla against the middle ear-13- This paper scale is applicable to the Chinese National Standard (CNS) A4 specification ( 210 X 297 mm) - ------------------------------- (Please read the notes on the back and fill out this page. 1297731 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed A7 ____ B7____· _ V. Invention Description (11) Inflammation and protection of young rats against Haemophilus influenzae type b bacteremia (Loosmore S. Μ· et al· 1998. Infect· Immun. 66: 899) The outer capsular sac (or large blister) has been isolated from H. influenzae (Loeb Μ·R·, Zachary A·L·, Smith D'H. 1981·J· Bacteriol. 145: 569-604; Stull TL, Mack K., Haas JE? Smit J.? Smith AL 1985. Anal. Biochem. 150: 471-480). Small pouch and blood-brain barrier penetration (Wiwpelwey B·, Hansen Ε· J·, S Cheld W. Μ· 1989 Infect. Immun· 57: 2559-2560), related to meningitis (Mustafa Μ. Μ·, Ramilo 0·, Syrogiannopoulos G· Α·, Olsen Κ, D·, McCraken GH Jr. , Hansen, EJ 1989. J. Infect. Dis. 159: 917-922) and related to DNA uptake (Concino M. F., Goodgal SH 1982 J. Bacteriol 152: 441-450). It can be shown that adhes and/or colonization factors can be present in large blisters through nasal mucosal epithelial binding and absorption (Harada T., Shimuzu T., Nishimoto K., Sakakura Y. 1989. Acta Otorhinolarygol. 246: 218-221). · An immune response to proteins present in the outer membrane sac has been observed in patients with various Haemophilus influenzae diseases (Sakakura Y., Harada T., Hamaguchi Y., Jin C. S. 1988. Acta Otorhinolarygol. Suppl (Stockh.) 454: 222-226; Harada T., Sakakura Y., Miyoshi Y. 1986. Rhinology 24: 61-66). Pseudomonas aeruginosa Pseudomonas contains Gram-negative, polarized flagella, a straight and slightly curled columnar body that grows under anaerobic conditions and does not form cells. Due to its limited metabolic requirements, Pseudomonas is ubiquitous and widely distributed in soil, air, sewage and plants. Many Pseudomonas, such as Pseudomonas aeruginosa, Class-14 - This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) -J.----------- Pack---- (Please read the notes on the back and fill out this page) I ^1 1 .1 Order --------- A7 B7

1297731 V. INSTRUCTIONS (12) Pseudomonas sinensis, Pseudomonas sinensis, Pseudomonas maltophilia and Pseudomonas syringae have been shown to be pathogenic to humans. In this column, the patina is considered to be an important human pathogen' because it is associated with the susceptibility of immunocompromised hosts and is responsible for the high mortality rate of inpatients. In the case of hospital infection caused by Pseudostellaria platensis, the main afflicted patients are those who receive long-term treatment and receive immunosuppressive agents, corticosteroids, anti-metabolic antibiotics or radiation. Pseudomonas, and in particular Pseudomonas aeruginosa, can produce a variety of toxins (such as blood lysin, fibrin, esterase, coagulase, citrate, internal and external toxins) causing the pathogens of these bacteria Sex. Furthermore, these organisms have high intrinsic resistance to antibiotics due to the presence of multiple drug efflux pumps. The latter feature often complicates the outcome of the disease. Due to the uncontrolled use of antibacterial chemotherapy, the frequency of hospital infections caused by patina has been greatly increased over the past three years. For example, in the United States, the economic burden of infection with P. aeruginosa hospitals is estimated to reach approximately US$4.5 trillion per year. Therefore, it is highly desirable to develop active or passive immunization vaccines against P. aeruginosa (for an overview see Stanislavsky et al. 1997. FEMS Microbiol. Lett 21: 243-277) Pseudomonas aeruginosa various combinations with bacteria and Secreted antigens have been the subject of vaccine development. Among the Pseudomonas antigens, the mucin-like substance, which is a bacterial mucus mainly composed of alginic acid, is found to be heterogeneous in terms of size and immunogenicity. The high molecular weight alginic acid component (30-300 kDa) appears to contain a reserving epitope, while the lower molecular weight alginic acid component (10-30 kDa) also has a reserving epitope outside the unique epitope. Among the proteins bound to the surface, PcrV, part of the type III secretion-indexing device, has been shown to be also immuno--15-this paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) •Γ. i J------------------r---book---------. (Please read the notes on the back and fill out this page. ) Ministry of Economic Affairs, Intellectual Property Bureau, Staff and Consumer Cooperatives, Printing 12,773,371 Ministry of Economic Affairs, Intellectual Property Office, Staff and Consumer Cooperatives, Printing A7 B7 V. Inventions (13) Interesting targets in vaccination (Sawa et al 1999 Nature Medicine 5: 392- 398) Antigens that are exposed to the surface include 〇_antigen (Lps 〇 特异 特异 特异 特异 特异 Η Η Η Η Η Η 特异 特异 特异 特异 特异 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 The chemical structure of the repeat unit in the Ο-specific polysaccharide has been described, and these data can be used to identify the 31 chemical forms of P. aeruginosa. A reserved epitope in all serotypes of P. aeruginosa, located in the core oligo vinegar and lipid raft region of LPS, and immunogens containing these epitopes can antagonize different P. aeruginosa immunotypes The immunity of the mice in the body is crossed and protected. Out of LPS is referred to as a ligand for Pseudomonas aeruginosa in combination with the respiratory and ocular epithelium of an animal. However, there is heterogeneity in this outer shell region between different serotypes. The epitope located on the inner shell is highly retentive and has proven to be accessible to the surface and is not obscured by the 〇-specific polysaccharide. To examine the protective properties of Ο Μ protein, a vaccine containing Pseudomonas aeruginosa 分子量 protein with a molecular weight of 20 to 1 〇〇 kDa was used in preclinical and clinical trials. This vaccine effectively antagonizes P. aeruginosa challenge in animal models and produces high levels of specific antibodies in human volunteers. Plasma from human volunteers contains anti-P. aeruginosa antibodies that provide passive protection and help restore recovery of 87% of patients with P. aeruginosa infection. Recently, a part of the outer membrane protein 〇prF is fused to (glutamic acid 19-83) and OprI (amino acid 21-83) (from Pseudomonas aeruginosa) to glutathione_S-transfer The fusion protein consisting of enzymes has been shown to protect old gas against 975 times 50% lethal dose of Pseudomonas aeruginosa (Knapp et al. 1999)

Vaccine. 17: 1663-1669) 0 -16- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) i —------··· (Please read the notes on the back and fill out this page) 1297731

Printed by the Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperatives V. INSTRUCTIONS (14) The present inventors have raised a number of related disadvantages for the above-mentioned wild-type large blister vaccine (whether naturally occurring or chemically prepared). Examples of this problem are as follows: There are immunogenic but variable proteins on large blisters (p〇tA; TbpB ' Opa [N. meningitidis b]; P2, p5 [uninhibited H. influenzae] ) This large blisters are only effective in antagonizing certain restricted bacterial species. The type-specificity of bacterial antibodies prevents this vaccine from being blocked in infants. - Non-protective (non-related) antigens present on large blisters (Rlnp, H8, ···)-antigens are the complement of the immune system - lack of important molecules that are adapted to produce (eg iron-regulated outer membrane proteins) , IROMP, s, expression mechanism of in vivo regulation) _ This condition is difficult to control in the production of large vesicles, making the amount of antigen on the surface more perfect - protective (especially reserved) low antigen expression level (NspA , P6) - LPS toxicity remaining on the surface of large blisters 'Because of the host-same structure, the possibility of autoimmune reaction (such as the capsular polysaccharide in Neisseria meningitidis serum B type, Neisseria) Lactose-N-neotetraose on LPS, the sugar structure in ntHi LPS 'glycan structure in cilia. Such problems hamper the use of large vesicular vaccines in human vaccine reagents. Especially in pediatric applications (< 4 years old), in which the responsiveness of the large vesicular vaccine is particularly important, and the large blister vaccine (as previously mentioned < the marketed MenB blister vaccine) has been shown to be immunized Protection is invalid -17- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) I -----r------------r--- -------- (Please read the note on the back and then fill out this page) 1297731 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 V. Invention description (彳5). Accordingly, the present invention proposes a method of using genetically engineered strains to alleviate the above problems, which results in an improved large blister vaccine. This method is particularly useful for the production of new vaccines against bacterial pathogens such as Neisseria meningitidis, Moraxella catarrhalis, Haemophilus influenzae, Pseudomonas aeruginosa and others. The large blister vaccine of the present invention has been designed to focus on the immunological response of some protective (preferably retaining) antigens or epitopes - which are harmonized in multiple reconstituted parts. The antigen is a complete 〇Mps, and the outer membrane sac of the large water cancer vaccine ensures its correct folding. The invention proposes a more perfect method for making OMP and LPS in the MV (large water cancer) vaccine, deleting the variable and non-protective 〇MPs with immunodominance, and deleting the variable region to generate retaining OMPs. , positively regulate the performance of protective 〇MPs, and eliminate the protective 〇Mps performance·^ controlled mechanism (such as iron restriction). Furthermore, the present invention proposes a method for reducing the toxicity of lipid A by modifying the lipid moiety or changing the composition of the phosphonium group while maintaining its auxiliary activity, or masking it. These new and improved methods each improve the large blister vaccine, however, more than one combination of these methods can be combined to produce the most complete genetically manipulated protein vaccine that is immunoprotective and non-toxic, particularly suitable for pediatric use. . SUMMARY OF THE INVENTION The present invention provides a genetically manipulated large blister preparation derived from Gram-negative bacteria, characterized in that the preparation is obtained by applying one or more of the following methods: a) reducing the immunological advantage of the large blister preparation Or a method without a protective antigen, the method comprising identifying the nature of the antigen, genetically manipulating the bacterial strain to produce less or no such antigen, and making a large blister from the strain; -18- The paper scale applies to Chinese national standards (CNS) A4 specification (21〇X 297 mm) — — — — — — i II l· III ^ --- — — — — — — (Please read the notes on the back and fill out this page) 1297731 A7 d V. INSTRUCTION DESCRIPTION (16b) - A method for positively regulating the expression of OMP antigens in a protective, endogenous (and preferably retentive) OMP antigen in a large blister preparation. This method includes following this antigen, genetic manipulation. The bacterial strain introduces a stronger promoter sequence upstream of the gene encoding the antigen, such that the gene is expressed at a higher level than in unmodified large blisters, and large blisters are prepared from the strain; c) a positive A method of modulating the performance of a protective (and preferably retained) 〇Mp antigen in a large blister preparation, which method comprises quantifying the antigen and genetically manipulating the strain to remove the repressive control mechanism of its expression (such as iron limitation), and the formation of large water marks in the strain; a method for modifying the lipid A portion of the bacterial LPS in the large water sore preparation, the method comprising identifying the lipid A portion of the LPS is toxic The gene, the genetically manipulated strain causes the gene to be reduced or cut off, and a large blister is made from the strain. · A method for modifying the lipid A portion of the bacterial LPS in a large blister preparation, the method comprising identifying UVS A lipid-partially less toxic gene's genetic manipulation of the strain so that a stronger promoter sequence can be introduced upstream of the gene, such that the gene is expressed at a higher level than in the unmodified control group, and from the strain a large blister; a method of reducing the toxicity of lipid A in a large blister preparation and increasing the level of protective antigen, the method comprising chromosomal genetic manipulation of the fine strain to be included in the coding a gene of a polymucin A peptide, or a derivative or analog thereof, fused to a protective antibody, and a large blister is prepared from the strain; I----.-----------h ---Order---------: (Please read the notes on the back and fill out this page) Printed by the Ministry of Economic Affairs, Intellectual Property Bureau, Staff and Consumers Co., Ltd. f) -19 - 1297731 A7 B7 V. Description of Invention ( 17) g) a method for preparing a retained 〇Mp antigen in a large blister preparation, the method comprising identifying the antigen, genetically manipulating the strain to delete a variable region of the gene encoding the antigen, and producing from the strain Large water cancer; h) - a method for reducing the performance of antigenic large blister preparations, which shares structural similarity with humans' and can induce autoimmune responses in humans (such as the sandwich polysaccharide of Neisseria meningitidis B) The method comprises identifying a gene involved in antigen synthesis, genetically manipulating the strain to reduce or sever the performance of the gene, and thereby forming a large blister in the strain; or i) one in a large water cancer preparation A protective, endogenous (and preferably retained) method for the positive regulation of OMP antigens, this method package This antigen is identified, genetic manipulation to introduce a strain to the antigen encoded in more than one gene copy number on the chromosome, which is heterogeneous and is a stronger promoter sequence control, blisters and prepared from this strain. A further aspect of the invention includes obtaining a segmentation process of the above-described large blister preparation 'including optimal positioning of a strong promoter to positively regulate the expression of antigens in the large blister, and differentially selecting antigens for forward and reverse regulation of various strains To make large blister preparations that are particularly suitable for vaccine applications. A preferred I week containing a large water cancer of the present invention is also proposed, which is particularly suitable for use as a vaccine to antagonize certain disease states. The carrier of the large blister of the present invention can be produced, and the modified strain of the large blister production of the present invention is also a further aspect of the present invention. The present invention is the first to propose a large blister vaccine which is immunoprotective and non-toxic when used in children under 4 years of age. BRIEF DESCRIPTION OF THE DRAWINGS -20- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) -L. i——--------l·——订------ --- (Please read the notes on the back and fill out this page) Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperatives Ministry of Printing and Economy Ministry Intellectual Property Bureau Staff Consumer Cooperatives Printed 1293731 A7 —--------B7____ V. Description of the invention (18) Figure 1: Reactivity of 735 mAbs in different colonies. Figure 2: Reactivity of specific monoclonal antibodies to whole cell Eusa. Figure 3: illustrates the pCMK vector used to deliver genes, operons and/or sputum in the genome of N. meningitidis. Figure 4: Analysis of the performance of PorA in total protein extracts of recombinant N. meningitidis serum type b (H44/76 derivatives). Total protein recovery

From cps- (columns 3 and 4), cps_porA::pCMK+ (columns 2 and 5) and cps-porA::nspA (columns 1 and 6) recombinant N. meningitidis serum b-type strain, and in SDS- Analysis was performed on a 12% polyacrylamide gel under PAGE conditions. The gel was stained with Coomassie blue (columns 1 to 3) or transferred to a nitrocellulose membrane and immunostained with anti-PorA monoclonal antibodies. Figure 5: Analysis of the performance of NspA in protein extracts of recombinant N. meningitidis serum type b strain (H44/76 derivative). Protein extraction from whole bacteria (columns 1 to 3) or outer membrane large blister preparations (columns 4 to 6), separation on SDS-P AGE on 12% acrylamide gel, and anti-accumulation using anti-- NspA multi-strain serum analysis. Analyze samples relative to cps_ (columns 1 and 6), cps_pora::pCMK+ (columns 3 and 4) and cps-porA::nspA (columns 2 and 5). Two types of NspA can be measured, one mature type (i8kDa) co-moved with the recombinant purified NspA, and the other shorter form (i5kDa). Figure 6: Analysis of the performance of D15/omp85 in recombinant B. meningitidis serum type b (H44/76 derivative) protein extract. The protein was extracted from the large blister preparation and separated on a 12% acrylamide gel by SDS-PAGE and analyzed by immunosuppression using anti-omp85 serum. Analysis is equivalent to cps· (2 columns) and cps-, PorA+, pCMK+Omp85/D15 (1 column) Recombinant-21 - This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) — 7 ------------------r---- ^--------- (Please read the notes on the back and fill out this page) 1297731 Ministry of Economics Property Bureau Staff Consumer Cooperatives Printed A7 B7 V. Inventive Notes (19) Samples of Neisseria meningitidis serum B strain. Figure 7: General strategy for delivery of regulatory gene expression by promoter (RS sites for restriction sites) Figure 8: Analysis by recombinant N. meningitidis serum type b cps-strain (H44/76 derivative) Outer membrane large blisters, proteins were extracted from the outer membrane large blister preparation and separated on a 4-20% gradient polyacrylamide gel under reducing conditions. The gel was stained with Coomassie Brilliant Blue R250. Columns 2, 4, and 6 correspond to 5 micrograms of total protein, while columns 3, 5, and 7 are labeled with 1 microgram of protein. Figure 9: Constructing a promoter to replace the plastid to positively regulate the performance/production of Omp85/D15 in N. meningitidis H44/76. Figure 10: Analysis of the performance of OMP85 in the total protein extract of recombinant NmB (H44/76 derivative). The gel was stained with Coomassie Blue (A) or transferred to a nitrocellulose membrane and immunostained with rabbit anti-〇MP85 (Neisseria gonorrhoeae) monoclonal antibody (B). Figure 11: Analysis of the performance of OMP85 in OMV preparations of recombinant NmB (H44/76 derivative). The gel was stained with Coomassie Blue (a) or transferred to a nitrocellulose membrane and immunostained with rabbit anti-OMP85 polyclonal antibody (B). Figure 12 illustrates the recombinant PCR strategy used to delete lac(R) in the chimeric porA/lac(R) promoter. Figure 13: Analysis of Hsf performance in total protein extracts of recombinant N. meningitidis serum type b (H44/76 derivatives). Total protein was recovered from Cps-PorA+ (column 1) and Cps-PorA+/Hsf (column 2) recombinant meningococcal nephrose halogen serum type B and coagulated with 12% polyacrylamide under SDS-PAGE conditions. Analysis on the gel. The gel was stained with Coomassie Blue. -22- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) - - -----r---- I — i II L----.. (Please read the back first Note: Please fill out this page again) 1297731 A7 V. INSTRUCTIONS (20) Figure 14: Analysis of GFP expression in total protein extracts of recombinant N. meningitidis (H44/76 derivatives). The total protein was recovered from Cps-P〇rA+ (column 1) and Cps-, P〇rA-GFP+ (columns 2 and 3) recombinant proteins, which were separated by PAGE-SDS on a 12% polyacrylamide gel. Then stained with Coomassie blue. Figure 15: Analysis by SDS-PAGE (Coomassie Blue staining) shows the pattern of major proteins on the surface of various recombinant large water sore preparations. Figure 16. Analysis of specific anti-Hsf responses of various large water cancer and recombinant large blister preparations using purified recombinant Hsf protein. Figure 17: Analysis of the performance of NspA in total protein extracts of recombinant NmB (serum B-type derivatives). The gel was stained with Coomassie Blue (A) or transferred to a nitrocellulose membrane and immunostained with mouse anti-P〇rA monoclonal antibody (B) or mouse anti-NspA# strain antibody (C). DESCRIPTION OF THE INVENTION The present invention relates to a combination of tools and methods useful for the manufacture of improved, genetically manipulated, large water cancer from Gram-negative bacterial strains. The invention includes methods for preparing recombinant large vesicles that are more immunogenic, less toxic and safer for use in human and/or animal vaccines. Furthermore, the present invention also describes the specific methods necessary for the purpose of vaccination, treatment and/or diagnosis for the purpose of constructing, controlling, obtaining and using recombinants derived from various Gram-negative bacteria, genetically manipulated large blisters. Using the method of the present invention, the biochemical composition of bacterial large blisters can be manipulated by acting/altering the expression of bacterial genes present in or associated with bacterial outer membrane large vesicles (outer membrane proteins or OMPs). Production of large water by means of deportation modification -23- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 public) (Please read the note on the back and fill in this page) 1297731

It is also within the scope of the present invention that the Ministry of Economic Affairs' Intellectual Property Office staff consumption cooperative prints sores to increase, decrease or adapt the performance of the genes encoding the outer membrane components. For the sake of clarification, the term "expressed" refers to all the inheritance necessary for the expression of a gene, or an operon, and the production and alignment of a comparable protein in the outer membrane of large water cancer, which is derived from a specific bacterial host. Essentials. The incomplete list of these features includes control elements (transcription and/or translation), protein coding regions, and alignment signals, which are present at appropriate intervals. Regarding the insertion of the promoter sequence, it is indicated in the present invention that it has at least a promoter function (sequence insertion, and preferably contains more than one other genetic regulation element in the expression 。. Further, the so-called "integration This refers to all the genetic elements necessary for the integration of DNA fragments in a particular bacterial host. These non-complete listings include delivery of vehicles (or vectors), recombination-containing regions, and screenable and counters. a filterable marker. Again, for clarification purposes, the so-called genetically manipulated strains produce less antigen" refers to any method that reduces the expression of key antigens compared to unmodified (that naturally occurring) large blister, so The performance is at least 10% lower, preferably at least 50% lower. ''A strong promoter sequence, which refers to a gene encoding a key antigen, and g, can increase the regulatory control of transcription." Positive regulation performance #曰Any method that enhances the performance of key antigens compared to unmodified (ie, naturally occurring) leeches. Understand that the amount of "positive regulation is based on The specific antigen changes, but does not exceed the amount that can disrupt the integrity of the large blister membrane. The positive regulation of the antigen refers to at least 丨〇% or more of the unmodified large water cancer. It is preferably at least 50% higher and better. At least 1% (more than 2 times). One aspect of the invention relates to the production of improved genetically manipulated large blister -24- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) - ------^--------------r---^ Order --------- (Please read the notes on the back and fill out this page) 1297731

, invention description (22 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing individual methods, the combination method of this method, and the large blister made by these methods: another aspect of the invention relates to the bacterial strain selected by genetic modification < a genetic tool for extracting improved and genetically manipulated large water cancer from the strain. The genetic manipulation steps of the methods of the invention can be carried out by various methods known to the artisan, for example, sequences (such as promoters or open reading) The architecture can be embedded: and the promoter/gene can be disrupted by factor-incorporating techniques. For example, in order to positively regulate the expression of a gene, a strong promoter can be inserted via a translocation factor of up to 2 to the upstream of the gene promoter codon (preferably Upstream 2〇〇_6〇〇bp, more ^约 upstream about 4〇〇bp). You can also use point mutation or deletion (especially in terms of gene regulation). However, this method may not be very Stable or uncertain, so genetic manipulation steps [especially as a), b), c), d), e), "and 丨" methods] are better through homogenous combination Preferably, the action occurs on at least 30 nucleotide nucleotide sequences (recombinant regions) on the bacterial chromosome, and at least 30 nucleotide sequences of the vector transformed in the strain (second recombination) Between the original regions). Preferably, the region has 4 (Mo〇〇 cores: acid. It is preferably 100-800 cores: y: acid, preferably 5 茫 nucleosides). The regions of the original recombination should be sufficiently similar, so that they can hybridize under highly urgent conditions (as will be later defined). Recombination can utilize a single recombination region on the chromosome and vector' or via a double transverse effect (Using 2 regions on chromosomes and vectors). For single recombination, the vector should be a circular dna molecule. For double recombination, the vector can be circular or linear-I ----^ ----! i!h — 丨—Book!! (Please read the notes on the back and fill out this page) -25 1297731 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau Employees Consumption Cooperative Printed 5, Inventions (23 ) DNA Molecules (see Figure 7). Better, apply dual recombination, The vector used is linear, as the modified strain thus produced is genetically stable. Preferably, the two recombined regions on the chromosome (and on the vector) are similar (compared to The same is the length, so as to promote the double effect. The dual traversal function allows two recombination regions on the chromosome (separated by the nucleotide sequence, χ, separate) and two on the carrier Recombination of the original region (by nucleotide sequence, gamma, separate) recombination, leaving unaltered chromosomes except for the interchange of X and gamma. The region with recombination can be located upstream or downstream of the key open reading architecture, or Two sides. These regions include both coding and non-coding sequences or a mixture of the two. The nature of χ and γ depends on the role of the desire, X can be all or part of the open reading architecture, and Υ has no nucleotides, which can cause the sequence χ to be deleted from the chromosome. In addition, Υ can be a strong promoter region to be embedded upstream of the open reading architecture, so X may not be nuclear-free at all. Suitable carriers in the compositions may vary depending on the type of recombination event to be performed' and the ultimate purpose of the recombination event. The integration vector used to deliver the sputum region can be an adaptable replication or suicide plastid, phage, transfer factor or linear DNA fragment obtained by restriction enzyme hydrolysis or PCR amplification. Recombination events can be selected using genetic markers that can be screened, such as genes that provide antibiotic resistance (such as oxymycin, erythromycin, chloramphenicol or gentamicin) to provide resistance to heavy metals and/or toxic compounds. The gene, or a gene that complements an auxotrophic mutation (such as pur, leu, met, aro). Variable and non-遂 immunodominant antigens Many surface antigens are variable between bacterial bacteria, and therefore only limitedly protected I l I -l·—ϋ I ϋ II 1_ ϋ I —mmm ϋ I ϋ ϋ ϋ ϋ ϋ · (Please read the notes on the back and fill out this page) 26- 1297731 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperatives Print A7 B7 V. Invention Description (24) Strain-related strains. A part of the present invention relates to the reduction or better deletion of a gene encoding a variable surface protein, resulting in a bacterial strain capable of producing a large water sore, which is administered once in a vaccine form. The potential is cross-reactive to antagonize various strains due to the higher effects exhibited by the intrinsic protein (retained in the outer membrane) on the volunteer's immune system. Examples of such variable antigens include: Neisseria gondii-Pile, which undergo antigenic changes, p〇rA, 〇pa, TbpB, FrpB; Haemophilus influenzae _P2, P5, cilia, :^八^protease; and Moraxella - CopB, OMP106. Other genotypes that can be reverse regulated or cleaved are genes that can be easily initiated (expressed) or cleaved by bacteria in vivo. As for the outer membrane protein encoded by this gene, it is not always present in bacteria. The presence of this protein in large vesicular preparations may also be due to the efficacy of the vaccine for the above reasons. A preferred example of reverse regulation or deletion is the Neisserial 〇pc protein. The anti-〇pc immunity produced by the Ope-containing blister vaccine has only effective protection because the infected organism can easily become 0pc_. Haemophilus influenzae HgpA and HgpB are other examples of this protein. " Yu Wanfa a), these variable or unprotected genes can be inversely regulated in performance, or eventually cut off. This has the surprising advantage described above, even if the immune system is concentrated on the preferred antigen, and the antigen is on the outer surface of the blister. Strains can be genetically manipulated in this manner by a variety of strategies, including translocation factors to disrupt the coding region or promoter region of the gene, or point mutations or deletions to achieve similar results. You can also use homogenous recombination to delete the L___ - 27 - Ϊ paper scale in the chromosome. Apply the Chinese National Standard (CNS) A4 specification (10) χ 297 public! ιί----------[ order--- ------- (Please read the notes on the back and then fill out this page) 1297731 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperatives Print A7 V. Inventions (25) Genes (where sequence X includes key gene parts) (preferably all) coding sequences). It can additionally be used to alter its strong promoter, which is replaced by a weaker (or a promoter) (wherein the acid sequence x includes a partial (preferably all) gene promoter region, and the nucleotide sequence γ Including weaker promoter regions [causing a decrease in key gene/operator performance], or no promoter region). In this case, recombination events occur better in the upper (7) (8) Qiu chromosome region of the key gene. Additional greening activity can be provided, resulting in the performance of key genes/operators (reverse regulation). This can be used for the performance of molecules that are toxic or not adequately supported by bacterial hosts. The protein is intact 〇Mps, and its variability is limited to one or a few of its surface exposure bands. Another aspect of the invention [Method g)] includes deletion of a DNA region that directs the synthesis of these surface exposed rings, which can result in The performance of a complete 0MP with an inherent surface exposure ring. The surface exposure of Haemophilus influenzae 2 and P 5 is a preferred example of a protein which can be transformed into the antigen of the parent fork reaction by this method. Again, homogenous recombination is the preferred method of performing this deportation. Method b)·Promoter delivery and language [姑:, 本=明 Further aspect relates to a method for modifying a large blister composition, which is to change the in situ control region of the key gene and/or operon expression. Modifications include the restriction of a key gene to express an endogenous promoter, or a replacement for all transcriptionally active agents. This different transcriptional activity may be provided by a variant of the endogenous control region (point mutation, deletion and/or incorporation), using a naturally occurring or modified heterologous promoter or a combination of both. The transcriptional activity provided by this change is better than that of the endogenous source - --------- Φ----i I, order--------- (please read the notes on the back first) Fill in this page) _ -28- Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1297731 A7 _____ B7 _ .____ V. Invention Description (26) (Introducing a stronger promoter) can cause key genes/operators Enhanced performance (positive regulation). This method is particularly useful for enhancing the production of immunologically relevant large water cancer components, such as outer membrane proteins and lipoproteins (preferably solid OMPs, usually present in low concentrations in large blisters).

Typical strong promoters that can be integrated into Neisseria are p〇rA [SEQ ID NO. 24], porB [SEQ ID NO: 26], lgtF, Opa, Pll〇, 1st and hpuAB. PorA and PorB are preferred prototrophic promoters. It has been determined (Example 9) that the PorB promoter activity is contained in the upstream nucleus of the porB promoter codon: y: a fragment of acid-1 to -250. In Moraxella, ompH, 〇mpG, ompE, ompBl, 〇mpB2, ompA, OMPCD and Ompl06 promoters are preferably used, and P2, P4, PI, P5 and P6 are better integrated in H. influenzae. child. The promoter is introduced into the 1000 bp upstream region using a preferred dual transverse homologous recombination technique, and the promoter can be placed anywhere 30-970 bp upstream of the promoter codon of the gene to be positively regulated. Although it is expedient for the promoter region to be highly relevant to the open reading architecture in order to have the most appropriate expression of the gene, the inventors have surprisingly found that promoter promoters can be used at a distance to generate a higher level of performance. increase. Therefore, the promoter is preferably inserted 200-600 bp upstream of the gene start codon, preferably 300-500 bp from the activated ATG, and preferably about 400 bp. 0 Method c)-Adapting the large blister component of the real estate to guide synthesis The performance of certain genes in certain large blister components can be carefully regulated. The composition of the components can be adjusted and adjusted according to various metabolic and/or environmental signals. Such signals include, for example, iron limits, oxygen and potential regulation, p Η and temperature changes, and nutritional changes. Adjustable real estate blister -29- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) Ί-r-------Aw ^-------Ί - -------- Awl (please read the note on the back and fill out this page) _ Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1297731 A7 _____Β7__ V. Invention Description (27) Some examples of components include Neisseria gonorrhoeae and iron-regulated extracellular membrane proteins (such as TbpB, LbpB), and receptor-expressable outer membrane proteins. The invention encompasses the use of the previously described genetic methods (Method a) or b)) to render the molecule as it is. In this way, the environmental signal that affects the expression of key genes can be overcome by the equivalent control region of the change/replacement gene, so that it becomes active in the original structure (such as deleting parts [better all] or inhibiting Control sequences, such as operon regions, or embedded promoters of strong promoter genes. For the iron-regulated gene, the fur operator can be removed. Alternatively, method i) can deliver additional sets of key genes/operators to the chromosome, which are artificially placed under the control of the original promoter. Methods (Π, and e) - LPS's shame 毒性 One of the biggest problems with the toxicity of large vesicle vaccines is the use of large vesicles in vaccines. A further aspect of the invention relates to a method of genetically detoxifying LPS in large blisters. Lipid A is the major component responsible for bacterial activation in LPS. Many of the mutations in genes involved in this pathway can cause phenotypes. However, the gene responsible for the terminal modification step may cause a temperature-sensitive (htrB) or random (msbB) phenotype. Mutations that cause reduced (or no-expression) performance of these genes can cause changes in lipid A toxicity. Indeed, the lipid-A of the non-laurel (htrB mutant) or the non-myristyl (msbB mutant) is less toxic than the wild-type lipid. Mutations in the lipid a 4,-kinase-encoding gene (ΙρχΚ) also reduce the toxicity of lipid a. Method d) is therefore related to the deletion of some (or preferably all) of the above one or more open reading architectures or promoters. In addition, the promoter can be replaced by a weaker promoter. Preferably, the homogenous recombination technique described above is used to carry out the process. ___ -30- ¥ Paper scale applicable to China National Standard (CNS) A4 specification (10) X 297 mm)-----

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1297731 V. INSTRUCTIONS (28) For this purpose, htrB&msbB gene sequences from N. meningitidis B, M. catarrhalis, and H. influenzae are additionally provided. The introduction of mutations in the polymyxin B resistance gene/region also alters the toxicity associated with the addition of amino arabinose to lipid A4, phosphate. These genes/regions may be pmrE, which encodes a UDP-glucose dehydrogenase' or a region of the anti-micropeptide-resistance gene shared by the Enterobacteriaceae family, which is related to the synthesis and transfer of amino arabinose. The gene pmrF, which is present in this region, encodes a polysterol-mannosyltransferase (Gunn js, Kheng, B. L., Krueger J., Kim K., Guo L., Hackett M., Miller S. · 1998· Mol. Microbiol. 27: 1171-1 182) Mutation in the PhoP-PhoQ regulatory system, a phosphoric acid-substituted two-component regulatory system (f·i· Ph〇P prokaryotic phenotype) , ph〇pc), or low Mg environment or culture conditions (ie activated phoP-PhoQ regulatory system) can cause the addition of amino arabinose to 4f-phosphoric acid and 2-hydroxy myristate-substituted myristic acid (musp Hydroxylation of acid). This modified lipid A exhibited reduced ability to stimulate E-selectin expression (from human endothelial bacteria) and tnF-alpha secretion (from human mononuclear bacteria). Method e) involves the forward regulation of these genes using the strategies described above. (Strong promoters are included, preferably using homogenous recombination techniques for this method). In addition, in addition to any of the mutations herein, the polymyxin B resistant strain may also be a vaccine-producing strain (in combination with one or more other methods of the present invention), since the large water cancer obtained from such a strain is also reduced. LPS toxicity (eg in meningococcal - van der Ley, P, Hamstra, HJ, Kramer, M, -31 - This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) - ϊ - — ^-------------r---订-------- (Please read the note on the back and fill out this page) Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative System 1277331 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention description (29)

Steeghs, L5 Petrov, A and Poolman, JT. 1994. In: Proceedings of the ninth international pathogenic Neisseria conference. The Guildhall, Winchester, England) o As for further changes (and further aspects of the invention), the inventors propose A method of attenuating a gram negative bacteria, the method comprising cultivating a strain in a growth medium containing 0.1 mg to 100 g of an amine-based indole sugar per liter of the medium. In a further aspect, a synthetic peptide mimicking polymyxin B (described below) binding activity can also be added to the large water sore preparation to reduce LPS toxicity (Rustici, A, Velucchi, M, Faggioni, R, Sironi, M) , Ghezzi, P, Quataert, S, Green, B and Porro M 1993. Science 259: 361-365; Velucchi, M, Rustici, A, Meazza, C, Villa, P, Ghezzi, P and Porro, M. 1997. J. Endotox. Res. 4:) o Method f) - Fixing homogenous salty heterogeneous proteins to adventitial large blisters while reducing the toxicity of LPS A further aspect of the invention relates to the use of polymyxin B peptides (or analogues thereof) The genetic sequence serves as a tool to align the fusion protein to the outer membrane. Polymyxin B is a cyclic peptide consisting of non-tRNA-encoded amino acids (produced by Gram-positive actinomycetes), which is closely related to the membranous A in the LPS of the outer membrane. Combine. This combination reduces the intrinsic toxicity (endotoxin activity) of LPS. Peptides which mimic the structure of polymyxin B and consist of a regular (encoded by tRNA) amino acid have been developed and which also bind to lipid A with strong affinity. These peptides can also be used to detoxify LPS. Among these peptides, referred to as SAEP-2 (terminal-Lys-Thr-Lys-Cys-Lys-Phe-Leu_Lys-Lys-Cys-terminus) has been shown to be very promising in this respect (Molecular Mapping and _ 32 _ ^ The paper scale applies to the China National Standard (CNS) A4 specification (210 297 297 Τ Τ ----U------------ l·------------- - (Please read the notes on the back and fill out this page) 1297731 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Print A7 _____ B7___ V. Inventions (30) detoxifying of the Lipid A binding site by synthetic peptides (1993)· Rustici , A·, Velucchi, M·, Faggioni, R·, Sir〇ni, M, Ghezzi, P·, Quataert, S·, Green, B· and M· P〇rro· Science 259, 361-365). The method of the invention f) proposes an improvement to this usage. It has been discovered that the DNA sequence (or derivative thereof) of the synthetic SEAP-2 peptide will be directed and genetically fused to a key gene (directing the synthesis of a T-adapted antigen or a protective antigen, which typically secretes toxins, cytosol or Periplasmic protein) can be used as a tool to target comparable recombinant proteins to the outer membrane of a preferred bacterial host (while reducing the toxicity of LPS). This system is suitable for applications of unstable proteins that cannot be directly exposed to the outside of large blisters. Thus, large blisters can be used as a delivery vehicle which exposes the protein to the immune system once the large blisters are inhaled by the T cells. In addition, genetic fusion should also include signal peptides or transmembrane regions such that the recombinant protein can be exposed to the host's immune system across the outer membrane. This strategy of targeting is particularly interesting in the case of genes encoding proteins that are not normally aligned with the outer membrane. This method can also be used to isolate recombinant large blisters that are enriched in key proteins. Preferably, the peptide is aligned with the target signal and can be used to add more than one major blister in the large blisters of the outer membrane, which is not found in a particular subcellular state under natural conditions. Non-exhaustive list of bacteria that can be used as a recipient host for this recombinant large water cancer include: Neisseria meningitidis, Neisseria gonorrhoeae, Moraxella catarrhalis, Haemophilus influenzae, patina Monocytogenes, Chlamydia trachomatis and Chlamydia pneumoniae. Although the gene of the construct is preferably genetically manipulated into the chromosome of the bacteria [Lei-33- This paper scale applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) (please read the notes on the back and fill in the form) Page) Install tr---------AVI. 1297731 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (31) Using method i)], another preferred embodiment is independently Recombinant proteins aligned to SAEP-2 were prepared and adhered to the large blister preparation at a later stage. A further specific example is the use of this construct in a protein purification process. This system can be used as part of a performance system to produce recombinant proteins. The SAEP-2 peptide marker can be used for affinity purification of proteins by adhering to a column containing solidified lipid A molecules. Method h) - Cross-reactive polysaccharides The isolation of bacterial outer membrane large vesicles from Gram-negative bacteria with capsules often results in the co-purification of capsular polysaccharides. In some instances, this "doped" substance may prove useful because the polysaccharide enhances the immune response provided by other large blister components. However, in other examples, it is doped in bacterial large blister preparations. Polysaccharides, which have been shown to be harmful in the use of large vesicles in vaccines, have been shown, for example, in at least in the case of N. meningitidis, the serum B group of the capsular polysaccharide does not provide protection immunity and is suspected to be in the human body. Inducing a harmful autoimmune response. Therefore, the method of the present invention is to genetically manipulate a bacterial strain to produce a large water cancer, so that it does not have Weidu. The large leeches will be applied to the human body. One of the large blister preparations A particularly preferred example is a group of N. meningitidis serogroup B, which has no capsular polysaccharide. This can be achieved by using a modified large blister producing strain for the production and/or delivery of a smiling membrane. The necessary genes have been destroyed. Let the English 2 = or the gene that is transported inactive... by mutation or point or correction in the control area, the coding area or both (preferably using the death and the same λ recombination technique). Capsular synthetic base Due to the loss of activity, it can also be achieved by using the over-expression of the anti-interest or the mutation of the conversion factor. Compared with the 34-paper standard, the national standard (CNS) A4 specification (21G 297 297 public H-----1- ---譬----------- -------- ί (Please read the notes on the back and fill out this page) -I n ϋ ϋ 1_1 nn · 1297731 Ministry of Economics Property Bureau Staff Consumer Cooperatives Print A7 V. INSTRUCTIONS (32) The best method is to delete some or all of the genes of N. meningitidis cps necessary for the synthesis and delivery of polysaccharides. For this purpose, replacement plastids can be used. PMF121 (described in Frosh et al. 199〇, M〇l 输1 4:121512 is called to deliver a mutation that has deleted the cpsCAD (+galE) gene. In addition, the siaD gene can be deleted, or reversed. Performance (the meningococcal siaD gene encodes a sialyltransferase, which is required for the synthesis of capsular polysaccharides and l〇s). This mutation also removes the structure of the sugar moiety of the bacterial Lps from the host. Supporting the old 1)-delivering a gene to the longitudinal group and further collecting the heterogeneous gene in the host chromosome An efficient strategy for controlling large water cancer preparation compositions is to deliver a set of DNA fragments containing more than one sputum to a Gram-negative bacterium, preferably a non-perfect list of preferred bacterial species. Among the recipients are Neisseria meningitidis, Neisseria gonorrhoeae, Moraxella catarrhalis, Haemophilus influenzae 'Pseudomonas aeruginosa, Chlamydia trachomatis, Chlamydia pneumoniae. It may be homogenous (or endogenous, naturally occurring in the genome of the manipulated bacteria) or heterogeneous (ie, not naturally present in the genome of the manipulated bacteria). Reintroduction may include unmodified A natural "promoter/gene/operator sequence or genetically manipulated 匣 where the promoter region and/or coding region or both have been altered. The incomplete list of preferred promoters that can be used for performance includes the following: p0rA from N. meningitidis or Neisseria gonorrhoeae, p〇rB, ibpB, tbpB, pllO, 1st, hpuAB, from influenza hemophilia P2, p5, p4, 〇mpF, pi, ompH, p6, hiii47, 〇 mpH from Moraxella catarrhalis, -35- This paper scale applies to China National Standard (CNS) A4 specification (210 χ 297 gong) PCT)

1^----------- — -----r------------ (Please read the notes on the back and fill out this page) __I Ministry of Economics Intellectual Property Bureau employee consumption cooperative printed 1297731 A7 __Β7 _ V. Description of invention (33) ompG, ompCD, ompE, ompBl, ompB2, ompA, λ pL, lac, tac, araB from E. coli or specifically for phage RNA polymerase Confirm the promoter, such as Ε· coli phage T 7 . The preferred gene in this system is a complete list of NspA, Omp85, PilQ, TbpA/B complexes, Hsf, PldA, HasR; Chlamydia MOMP, HMWP; Moraxella OMP106, HasR, PilQ, OMP85, PldA; FHA, PRN, PT of Bordetella pertussis. In a preferred embodiment of the invention, the expression is achieved by homologous and/or position-specific recombination, delivery and integration into bacterial chromosomes. The integration vector used to deliver the gene and/or operon can be a splicing or suicide plastid, phage, trans factor or linear DNA fragment obtained by restriction enzyme hydrolysis or PCR amplification. The better integration is to align the chromosomal regions that are absent for in vitro growth. The preferred regions that can be used to align DNA integration include: N. meningitidis and porA, porB, opa, opc, rmp, omp26, lecA, cps, lgtB genes, NTHi of N. meningitidis PI, P5, hmwl/2, IgA-protease, fimE gene; LecAl, lecA2, ompl06, uspAl, uspA2 gene of Moraxella catarrhalis. In addition, the performance of the components used to regulate the large water sore component can be delivered to the preferred bacteria by using the vector of the gene accessory, such as a circular/linear replica plastid, a plastid, a phage, and a lysate. A prophage or bacterial artificial chromosome. Recombination events can be selected using genetic markers that can be screened, such as antibiotic resistance genes (such as oxytetracycline, erythromycin, oxytetracycline or gentamicin) to provide resistance to heavy metals and/or toxic compounds. Gene, or a gene that complements auxotrophic mutations (eg pur, leu, met, aro). -36- This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) I;---h I----------- (Please read the notes on the back and fill in This page) order--------Λ 1297731 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (34) Outer membrane big water disease, outer membrane bacteria large water cancer represents a pole An attractive system that produces gas and separates and delivers heavy, tissue proteins for use in vaccines, treatments, and/or diagnostics. Further aspects of the invention are related to foreign, heterogeneous protein to outer membrane performance. And the purpose of targeting, and the use of bacteria to produce recombinant large water cancer. A preferred method of achieving this is via a method comprising the step of introducing a heterogeneous gene which is controlled by a strong promoter sequence and introduced into the chromosome of the Gram-negative strain via homologous recombination. Large blisters can be made from the modified strains produced. In the production of recombinant large blisters, the bacteria that can be used as recipients of the host are not fully listed, including: Neisseria meningitidis, Neisseria gonorrhoeae, Moraxella catarrhalis, Haemophilus influenzae, patina Monocytogenes, Chlamydia trachomatis, Chlamydia pneumoniae. The genes expressed in this system may be derived from viruses, bacteria, halogenated parasites or higher nucleus cells. The application of the present invention includes the expression of M. catarrhalis, Haemophilus and/or Pseudomonas outer membrane proteins (complete, multi-argument and/or lipid) in recombinant N. meningitidis large blisters protein). Preferably, the integrated site is as described above, and the gene which can be preferably introduced is a protective antagonist in the bacteria from which it is isolated. Preferred protective genes for each bacterium are as follows. Further preferred applications are: large blisters produced from modified Haemophilus influenzae, wherein the heterogeneous gene is a protective OMP from Moraxella catarrhalis, and is produced by modified Moraxella catarrhalis The big blisters, the heterogeneous gene in P is the protective OMP from H. influenzae (for the gene embedded car six I pen - set---------) (please read the back of the note first) Matters fill out this page) -37- 1297731 Ministry of Economic Affairs Intellectual Property Bureau Η Consumer Cooperatives Print A7 V. Invention Description (35 good areas as shown above, and better protective antigens as described below). It is caused by the prevention or / or oral treatment of sexually transmitted diseases (STDs). It is more difficult for participants to decide whether the main cause is infection with T. coli or Chlamydia trachomatis. These two organisms are salpingitis. This disease can cause infertility in the host. Therefore, if STD can effectively vaccinate or vaccinate the combination vaccine of this organism as a cause of disease, it will be very useful. The main outer membrane of Chlamydia trachomatis has been shown. Protein (MOMP) is guaranteed The standard of sexual antibodies. However, the structural integrity of this intact membrane protein is very important in the introduction of this antibody. In addition, the epitopes confirmed for these antibodies are variable, and more than one serotype is defined. As described earlier in the present invention, more than one membrane protein can be appropriately folded in a large blister outer membrane preparation. The outer membrane can exhibit multiple serozoin MOMP serovar variants of the bacterium, which is genetically manipulated and produced by Large vesicles provide a single solution to the correct folding of membrane proteins, protection against the presentation of various serum variants of M0Mp serotypes, and simultaneous prevention/treatment of gonococcal infections (so participants) It is not necessary to first decide which organism will cause a specific clinical symptom. Both organisms can be vaccinated at the same time, so that the treatment of STD can be performed at an early stage.) The best place for gene insertion in the chromosome of gonorrhea is shown in The other preferred protective gauline protoplast genes that can be included are HMWP, PmpG and disclosed in WO 99/28475. 〇MPs. Heterogeneous proteins are aligned to the outer membrane big blisters·· The performance of some heterogeneous proteins in bacterial blisters requires the addition of a signal to the outer membrane. The best way to solve this problem is in heterogeneous genes and -38- Table paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) II----r----------- (Please read the notes on the back and fill in the form) Page) Order---------. Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1297731 A7 __ B7_ V. Inventive Note (36) Genetically fused between the genes of the resident OMP, as a recombinant protein A specific pathway to the big blisters. Preferably, the heterogeneous gene is a signal peptide sequence fused to this OMP. Neisseria hominis preparations, when carried out on Neisseria, include gonorrhea and meningococcus (especially Neisseria meningitidis type B), more than one of the following genes (encoding protective antigens) It is preferred by method b) and/or i) for forward regulation: NspA (WO 96/29412), Hsf-class (W0 99/31132), Hap (PCT/EP99/02766), PorA, PorB, OMP85 (WO 00/23595), PilQ (PCT/EP99/03603), PldA (PCT/EP99/06718), FrpB (WO 96/31618), TbpA (US 5,912,336), TpbB, FrpA/FrpC (WO 92/01460 ), LbpA/LbpB (PCT/EP98/05117), FhaB (WO 98/02547), HasR (PCT/EP99/05989), lipo02 (PCT/EP99/08315), Tbp2 (WO 99/57280), MltA (WO 99/57280), and ctrA (PCT/E00/00135). It is also preferred to introduce genes of other Gram-negative bacteria heterogeneously. In the case of reverse regulation of method a), it is preferred to use more than one of the following genes: PorA, PorB, PilC, TbpA, TbpB, LbpA, LbpB, Opa and Ope ° by method d) in the case of reverse regulation, with the following More than one gene is preferred: htrB, msbB and ΙρχΚ. In the case of forward regulation via method e), more than one of the following genes is used: pmrA, pmrB, pmrE and pmrF. For method c), the preferred control sequences are: fur operator region (especially for either or both TbpB or LbpB genes); and DtxR manipulation -39- This paper scale applies to Chinese national standards (CNS) )A4 size (210 X 297 mm) -I ------------------- (Please read the notes on the back and fill out this page) Order----- ---- 1297731 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention Description (37) Sub-area. For the reverse regulation via method h), one or more of the following genes are preferred: galE, siaA, siaB, siaC, siaD, ctrA, ctrB, ctrC and ctrD. Pseudomonas aeruginosa large water disease preparation When the method b) and/or i) is positively regulated, one or more of the following genes (encoding a protective antigen) are preferably PrcV, OprF, OprI. It is also a preferred gene for heterologous introduction of other Gram-negative bacteria. When the Moraxella catarrhalis large blister preparation is positively regulated by methods b) and/or i), one or more of the following genes (encoding a protective antigen) are preferred: OMP106 (W0 97/41731 &amp; W0 96/34960 ), HasR (PCT/EP99/03824), PilQ (PCT/EP99/03823), OMP85 (PCT/EP00/01468), lipo06 (GB 9917977.2), lipolO (GB 9918208.1), lipoll (GB 9918302.2), lipol8 (GB 9918038.2), P6 (PCT/EP99/03038), ompCD, CopB (Helminen ME, et al (1993) Infect. Immun. 61:2003-2010), D15 (PCT/EP99/03822), OmplAl (PCT/EP99/ 06781), Hly3 (PCT/EP99/03257), LbpA and LbpB (WO 98/55606), TbpA and TbpB (WO 97/13785 &amp; WO 97/32980), OmpE, UspAl and UspA2 (WO 93/03761), And Omp21. It is also a preferred gene for the heterologous introduction of other Gram-negative bacteria. For the reverse regulation via method a), one or more of the following genes are car-payers: CopB, OMP106, OmpBl, TbpA, TbpB, LbpA and LbpB 〇 for the reverse regulation via method d), one or more of the following The genes are better · htrB, msbB and ΙρχΚ. -40- This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm), .J—-------Aw s--------1—.—Αν. (Please read the notes on the back and fill out this page) _ 1297731 Ministry of Economic Affairs Intellectual Property Bureau Employees Consumption Cooperative Printed A7 B7 V. Description of Invention (38) For positive regulation via method e), one or more of the following genes are Preferred: pmrA, pmrB, pmrE and pmrF 嗜H. influenzae large blister preparations For the positive regulation via method b) and/or i), one or more of the following genes (encoding protective antigens) are preferred: D15 (WO 94/12641), P6 (EP 281673), TbpA, TbpB, P2, P5 (WO 94/26304), OMP26 (WO 97/01638), HMW1, HMW2, HMW3, HMW4, Hia, Hsf, Hap, Hin47, and Hif (genes in this operon should be positively regulated to positively regulate cilia). It is also a preferred gene when heterologously introduced into other Gram-negative bacteria. For reverse regulation via method a), one or more of the following genes are preferred: P2, P5, Hif, IgAl-peptone per HgpA, HgpB, MHW1, HMW2, Hxu, TbpA, and TbpB. In terms of method d) reverse regulation, one or more of the following genes are preferred: htrB, msbB and ΙρχΚ. By way of method e) forward regulation, one or more of the following genes are preferred: pmrA, pmrB, pmrE and pmrF 〇 vaccine blends. A preferred embodiment of the present invention is a blend of the large blister preparation of the present invention in a vaccine, wherein Included are pharmaceutically acceptable excipients. The large blister preparation is prepared from any of the above modified strains and can be achieved by any method well known to the artisan. The method disclosed in EP 301992, US 5,597, 572, £ 1 1243 or 1184, 271, 147 can be used, and preferably, the method described in Example 8 is used. -41 - This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) i*!---L------------- (Please read the notes on the back first) <br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br> Eds Powell MF &amp; Newman MJ) (1995)

Plenum Press New York) The large blister preparation of the present invention can be adjuvanted with the vaccine blend of the present invention. Suitable adjuvants include aluminum salts such as aluminum hydroxide gel (aluminum) or aluminum phosphate, but may also be calcium (especially calcium carbonate), iron or zinc salts, or may also be deuterated tyrosine. , or a deuterated sugar, a cationically or anionically derivatized polysaccharide or an insoluble suspension of polyphosphene. Suitable Thl adjuvants which may be used include: monophospholipid sulfhydryl lipid a, in particular 疋3-de-〇-deuterated monophosphonium lipid A, and monophosphoryl lipid a, preferably 3 - go _ 〇 醯 醯 单 monophosphoryl lipid A (3D_MpL) plus a combination of aluminum salts. The enhanced system involves a combination of monophosphoryl lipid A and a capsaicin derivative, particularly a combination of QS21 and 3D-MPL, as disclosed in w〇94/〇〇153, or a less reactive composition, Among them, QS21 is quenched with cholesterol as disclosed in WO 96/33739. A particularly potent adjuvant blend is QS21 3D-MPL and a tocopherol in an oil/water emulsion, as described in w〇 95/1721®, and is a preferred blend. The vaccine may contain halogen, preferably QS21. It may also contain oil/water emulsions and tocopherols. Unmethylated CpG containing an oligonucleotide (w〇 96/02555) is also a preferred hair coloring agent for the TH1 reaction and is suitable for the present invention. The vaccine formulation of the present invention is administered via the systemic or mucosal route and can be used to protect or treat a mammal suspected of infection. These modes of administration include injection by intramuscular, intraperitoneal, intradermal or subcutaneous routes; or via mucosal administration to the mouth/digestive tract, respiratory tract, and genitourinary tract. Therefore, the one-42- paper size of the present invention is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm). (-Please read the back note first and then fill in this page) tr----- ----Awi. 1297731 Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperative, Printed A7 V. Invention Description (4〇) The method is to immunize a human host to antagonize the disease caused by Gram-negative bacteria. The method comprises administering to the host an immunoprotective dose: a large blister preparation of the invention. The amount of antigen in each vaccine formulation is selected to be an immunosuppressive response and to have no significant side effects in a typical vaccinator. This dose will vary depending on the particular immunogen used and how it is presented. In general, T, each dose can be expected to contain 丨_丨〇 〇 micrograms of protein antigen, which is 5-50 micrograms, more preferably 5 _25 micrograms. The most appropriate dose for a particular vaccine can be determined by standard studies, including observation of a suitable immune response in an individual. After initial immunization, the individual receives more than one dose of booster immunization at moderate intervals. Ghost cells or killed whole cell vaccines The inventors contemplate that the above-described improved methods for large blister preparations and vaccines can be extended to ghost cells or killed whole cell preparations and vaccines (with the same advantages). The modified Gram-negative strains of the present invention for the preparation of large blister preparations can also be used to make ghost cells and killed whole cell preparations. Methods for making ghost cell preparations (empty cells with intact envelopes) from the gram-negative bacteria are well known in the art (see, for example, WO 92/01791). Methods for killing whole cells to make inactive cell preparations that are available for use in the past are also well known. The so-called "large water cancer preparation" and "big blister vaccine" and the methods described in this document can be applied to &quot;ghost cell preparation" and "ghost cell vaccine" and "killed whole cell preparation π and 'kill" respectively. Dead whole cell vaccine" for the purposes of the present invention. The combination can be used to produce modified strains using more than one of the above methods, 43- to the paper size applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ------- 111111---- -r-------1111 (Please read the note on the back and fill out this page) 1297731 A7 V. INSTRUCTION DESCRIPTION (41) The improved large blister preparation of the present invention is thus reconstituted. It is preferred to use this method: more preferably - more than 2 (3, 4, 5, 6, 7, 8, or 9) to prepare a large blister vaccine. Since each additional method is used in the manufacture of large blister vaccines, the improved methods are combined with other methods to produce the best genetically manipulated large water cancer preparations. Preferred meningococcal (especially N. meningitidis type B) large blister formulations, including methods a), b), d) and/or e), and h). This large blister preparation is safe (no similar to the host structure), non-toxic, and such a configuration allows the host immune response to focus on a high level of protective (and preferably retentive) antigen. The above elements are integrated to produce the most suitable large water cancer vaccine. For the same applies to Moraxella catarrhalis and unformed H. influenzae, preferred large blister formulations include methods a), b) and d) and/or e). A further aspect of the invention is a whole cell vaccine for pediatrics that is immunoprotective and non-toxic to Gram-negative large water cancer&apos; ghost cells or killed. The so-called pediatric application is for infants under 4 years of age. Immunoprotective means that at least 40% (and preferably 50, 60, 70, 80, 90 and 100%) of the infants are seroconvertible (the bacteriostatic activity is increased by a factor of 4 [5%% of the antisera dilutions when the bacteria die) - See, for example, PCT/EP98/05117) to antagonize one of the heterogeneous strains selected from the predominantly pure elites known. For meningococcal type B, these strains differ from the large blister-producing strains in P〇rA type, and should preferably be 2, 3, 4 or better, all 5 strains: H44/76, M97/ 252078, BZ10, NGP165 and CU385. For H. influenzae that cannot be classified, these strains are preferably 2, 3, 4 or the last 5 of all • 44- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ) J---------------- (Please read the notes on the back and fill out this page) ·1111111

1297731 V. INSTRUCTIONS (42) Strains: 3224A ' 3219C ' 3241A, 640645 and A840177. For Moraxella catarrhalis, the strain should preferably be 2, 3, 4 or preferably 5 of all = strains ATCC 43617, 14, 358, 216 and 2926. Non-toxicity indicates that the activity of the known LAL and pyrogenicity analysis shows that the activity of the endogenous hormone is significantly reduced (2 - 4 times, preferably 1 〇). Vaccine Combinations A further aspect of the invention is a vaccine combination comprising a large blister preparation of the invention, plus other antigens which are usefully useful for antagonizing certain diseases. It has been found that large water sores are particularly suitable for reconciling with other antigens as they may be beneficial for the mixed antigens. In a preferred combination, the meningococcal B-type large blister preparation of the present invention is conjugated to 1, 2, 3 or preferably all of the following meningococcal capsular polysaccharides, either pure or yoke thereof As a protein carrier: A, C, γ or W. This vaccine can be beneficially used as a general meningococcal vaccine. In addition to the use of the meningococcal type B large blister preparation of the present invention, it is also conceivable that the blend may additionally contain two or more (preferably several) strains belonging to different subtypes/serotypes (e.g., selected from P1.15, Wild type meningococcal sputum type large blister preparations of P1.7, 16, Ρ1·4 and Ρ1·2). In a further preferred embodiment, the meningococcal sputum-type large blister preparation of the present invention [or a mixture of two or more wild type meningococcal β-form preparations described above] is preferably blended with 1, 2, 3 or 4 Simple or conjugated meningococcal capsular polysaccharide A, C, Υ or W, tunable to conjugated H. influenzae type b capsular polysaccharide, and more than one simple or conjugated pneumococcal capsule Polysaccharide. Depending on the requirements, the vaccine may also contain more than one protectable sink-45- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) -丨j---h------- -------r---订--------- C Please read the notes on the back and then fill out this page} Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperatives Printed Economy Ministry Intellectual Property Bureau employees Consumer Cooperatives Printed 1229731 A7 ————_B7__ V. Description of Invention (43) Primary antagonistic antigen of Streptococcus pneumoniae. This vaccine can be beneficially used as a general meningitis vaccine. The A. aureus capsular polysaccharide antigen is preferably selected from the following serotypes: 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A , 19F, 20, 22F, 23F and 33F (preferably selected from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F). Preferred pneumococcal protein antigens are pneumococcal proteins exposed to the outer surface of pneumococci (which may be confirmed by the host's immune system at least during the life cycle of the pneumococcal part), or may be proteins secreted or released by pneumococci . Preferably, the protein is a toxin, an adhesive, a 2_component signal transducer, or a lipoprotein of Streptococcus pneumoniae, or a fragment thereof. Particularly good proteins include the following: pneumonia lysin (preferably chemically treated or mutated to attenuate) [Mitchell et al. Nucleic Acids Res. 1990 Jul 11; 18 (13): 4010 &quot;Comparison of pneumolysin genes and proteins from Streptococcus pneumoniae types 1 and 2·'', Mitchell et al. Biochim Biophys Acta 1989 Jan 23; 1007 (1): 67-72 &quot;Expression of the pneumolysin gene in Escherichia coli: rapid purification and biological properties·,丨,WO 96/05859 (A· Cyanamid), WO 90/06951 (Paton et al), WO 99/03884 (NAVA)]; PspA and its transmembrane deletion variant (US 5804193-Briles et al.); PspC and its wearing Membrane deletion variant (WO 97/09994-Briles et al); PsaA and its transmembrane deletion variant (Berry &amp; Paton, Infect Immun 1996 Dec; 64 (12): 5255-62 &quot;Sequence heterogeneity of PsaA,a 37-kilodalton putative adhesin essential for virulence of Streptococcus pneumoniae"); pneumococcal test _46_ This paper scale applies to Chinese National Standard (CNS) A4 specification (210 x 297 mm) II---------- ------------Book --- ------ (Please read the notes on the back and fill out this page) 1297731 A7 __B7_ V. Inventions (44) (Please read the notes on the back and fill out this page) Combine proteins and their membranes to remove changes Type; CbpA and its transmembrane deletion variant (WO 97/41151; W0 99/51266); glyceraldehyde-3-phosphate-dehydrogenase (Infect. Immun. 1996 64:3544); HSP70 (WO 96/40928) PcpA (Sanchez-Beato et al. FEMS Microbiol Lett 1998, 164: 207-14); class-M protein, SB Patent No. EP 0837130; and viscosin 18627, SB Patent No. EP 0834568. Further preferred pneumococcal protein antigens are disclosed in WO 98/18931, in particular selected from WO 98/18930 and PCT/US99/30390. In still further preferred embodiments, the Moraxella catarrhalis blister preparation of the present invention is tunable with more than one simple or conjugated pneumococcal capsular polysaccharide, and one or more flu-insensitive sera that protects the host against untyped The antigen of blood bacteria infection. The vaccine may also contain more than one protein antigen that protects the host against S. pneumoniae infection, as desired. The vaccine may also contain more than one antigen that protects the host against RSV and/or one or more protects the host against the influenza virus. This vaccine can also be beneficially used as a general otitis media vaccine. The Department of Economic Intelligence's Intellectual Property Office staff consumer cooperative prints better unswappable H. influenzae protein antigens, including pilin protein (US 5766608) and peptide-containing fusions (such as LB1 fusions) (US 5843464-Ohio State) Research Foundation), OMP26, P6, protein D 'TbpA, TbpB, Hia, Hmwl, Hmw2, Hap and D15. Preferred influenza virus antigens include intact, live or inactive viruses, dividing influenza viruses, growing in egg or MDCK cells, or Vero cells or intact influenza virions (eg, R. Gluck, Vaccine, 1992, 10). , 915-920) or purified or recombinant protein, such as HA, NP, 47- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1297731 A7 Ministry of Economic Affairs Intellectual Property Bureau employees Printed by the Consumer Cooperatives. V. INSTRUCTIONS (45) NA or Μ protein, or a combination thereof. Preferred RSV (breathing sputum fusion virus) antigens include F glycoprotein, (10) protein, H Ν protein, or derivatives thereof. In still another specific example, the untypeable influenza virus squid preparation of the present invention can be adjusted with more than one simple or common pneumococcal capsular polysaccharide, and one or more protects the host against antagonism of Moraxella catarrhalis Infected antigen. Optionally, the vaccine may also contain more than one protein antigen that protects the host against S. pneumoniae infection. The vaccine may also contain, as desired, an antigen that protects the host against RSV, and/or more than one antigen that protects the host against the influenza virus. This vaccine can be beneficially used as a general otitis media vaccine. &amp; Nucleic Acids of the Invention A further aspect of the invention relates to the proposed novel nucleotide sequences which can be used in the methods of the invention. A special region upstream of each gene in each strain is proposed, which can be applied to methods such as small methods and other methods. A general method for analyzing two regions of non-coding bacterial genes, and their use in the development of gene regulation in large water cancers, develop non-coding two-sided regions of specific genes, which contain regulatory elements important for gene expression. This regulation occurs both above the transcription and translation layers. Attacking these regions &lt; sequences, whether upstream or downstream of the open reading architecture of the gene, can be obtained from the DNA sequence. This sequence data will determine possible regulatory features, such as different promoter elements, terminator sequences, sequence elements that can be guided, suppressors, elements responsible for phase changes, S, D sequences, and I*-- --------I -----r---订--------- (Please read the notes on the back and fill out this page) 48- Ministry of Economic Affairs Intellectual Property Office staff consumption Cooperatives print 1297731 V. Invention descriptions (bright) and areas of potential secondary structure for regulation, as well as other regulatory features or sequence patterns. This sequence data can be used to regulate the natural performance of the genes in question. Gene expression, forward regulation can be accomplished by altering the promoter, the s_d sequence, potential suppressor or operator requirements, or other requirements. In addition, the reverse regulation of performance can be achieved by a modified version of the class. In addition, by altering the sequence of phase changes, the expression of the gene can be placed under the control of phase changes or unraveled from this regulation. In another approach, the performance of the gene can be placed under the control of more than one inducible element of regulatable performance. Examples of such regulation include, but are not limited to, induction by temperature shift, addition of an inducer, such as selected carbohydrates or derivatives thereof, trace elements, vitamins, cofactors, metal ions, and the like. Such modifications as described above can be introduced in a number of different ways. Sequences involved in the expression of a gene can be modified in vivo by any mutation, followed by the choice of the desired phenotype. Another approach involves the isolation of key regions and modification by any mutation, or displacement of position-instructions, insertion or deletion of mutations. The modified region is then introduced into the bacterial gene by homologous recombination and evaluated for its effect on gene expression. In another approach, sequence information from the region of interest can be used to replace or delete all or part of the natural regulatory sequences. In this case, the aligned regulatory regions are isolated and modified, thus containing regulatory elements from another gene, regulatory elements from different genes, synthetic regulatory regions, or any other regulatory region, or deletion of wild-type Selected portions of the regulatory sequence. These modified sequences can be reintroduced into the genome of the bacteria via homologous recombination. -49- This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) I- . -----r------------ (Please read the back of the note first) 1277531 A7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (47) 矣 method b), gene expression can be controlled by replacing its promoter with a strong promoter (via The upstream sequence of the gene is isolated, the in-line modification of this sequence, and reintroduction into the genome by homologous recombination). Positive regulatory performance is obtained in both the bacteria and the outer membrane capsules obtained (or made) by the bacteria. In another preferred embodiment, the pathway can be used to produce recombinant strains with improved characteristics for vaccine applications, as described above. These include, but are not limited to, attenuated strains, strains with increased expression of selected antigens, (3) strains in which genes that interfere with immune responses are knocked down (or reduced in performance), and proteins with immunological advantages are subject to Controlled strains. SEQ ID NOs: 2-23 '25, 27-38 are the upstream sequences of N. serovar (upstream of the start codons of various preferred genes), each containing about 1 〇〇〇 (9). SEQ ID NO. 39-62 contains about 10 bp of the upstream sequence of M. catarrhalis (upstream of each preferred gene). SEQ ID NOs: 63-75 are upstream sequences of H. influenzae (upstream of the start codons of each preferred gene) each containing about 1000 bp. All of these can be used in genetic methods (especially homogenous recombination), linked to up-regulation or reverse regulation (as in the open reading architecture above. SEQ ID N〇: 76_81 is from Neisseria) , Moraxella catarrhalis, and the coding region of the HtrB and MsbB genes of Haemophilus influenzae. These can be used in genetic methods (especially homogenous recombination) to reverse the enthalpy (especially deletion) of Shao (good) Is all) these genes [method d)]. Another aspect of the invention is an isolated polynucleotide sequence which, under high stringency conditions, is capable of binding to at least 30 of 10 nucleofenacs of SEQ ID NOs: 2-23, 25, 27-81 or Complementary strands, hybrids. Preferably, the separated sequence should have enough J---------------^---------- (Please read the notes on the back and fill out this page. ) -50- 1297731 A7 B7 V. INSTRUCTIONS (48) (Please read the notes on the back and fill out this page) The length is sufficient to recombine with the chromosomal sequence, if it is a suitable carrier. One part - 3 甞 one nucleotide acid (preferably 4 〇, 5 〇, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nucleic acids). Preferably, the isolated polynuclear acid: the acid should contain SEq ID NO: 2-23, 25, 27-81 of at least 30 nucleotides (preferably at least 4, 50, 60, 70, 80, 90, 100) , 200,300 '400 or 500 nucleotides) or its complement. Highly urgent conditions used in this way include, for example, 6X ssc, 5χ Denhardt '0.5% SDS and 100 μg/ml fragmented and denatured salmon sperm DNA, hybridized at 65X: overnight, then 2X SSC, 0.1% SDS Wash it at room temperature for about once! 〇 min, followed by 65t for about 15 minutes, followed by at least 2 5 minutes at room temperature in 〇·2χ SCC ’ 0.1% SDS. The Ministry of Economic Affairs, Intellectual Property Office, and the Consumer Cooperatives Printing Department further uses the isolated polynucleotide sequence of the present invention to carry out genetic engineering within 1 bp upstream of the Gram-negative chromosomal gene (eg, embedding factor) Or position-specific mutations or deletions, but preferably homogenous, and acting) to increase or decrease the performance of the gene. The strain 'which is better to undergo recombination' is the same as the strain which obtains the upstream sequence of the present invention. However, meningococcal bacteria A, B, C, Y and w and the gonococcus gonorrhoeae genomes are sufficiently similar that the upstream sequences of these strains are suitable for designing into vectors for such engineering in other strains. This is also the case for H. influenzae and H. influenzae that cannot be classified. EXAMPLES The following examples were performed using standard techniques and are well known and routinely performed by the art unless otherwise specified. The examples are for illustrative purposes only and are not limiting. Example 1: Recording a serotype strain of N. meningitidis b lacking pen sugar • 51 - Not Am again for weekly use of hyperthyroidism; f indicating (CNS) A4 size (21〇 X 297 mm) 1297731

Printed by the Ministry of Economic Affairs, Intellectual Property Office, and the Consumer Cooperatives. The plastid PMF121 (Frosch et al. 199〇) was used to construct the N. meningitidis B strain lacking the capsular polysaccharide. This plastid contains a two-sided region or a gene encoding the gene of the B group polysaccharide (B PS) biosynthetic pathway. The deletion of B PS can result in the loss of B group capsular polysaccharide expression, and the deletion of gam activity sets can result in the synthesis of galactose-deficient Lps. Transformation of the strain: Neisseria meningitidis B H44/76 strain (b: 15: p17, 16; L〇s 3, 7, 9) was selected for transformation. On the sMH plate (without erythromycin) on the cells [ο] After one night of incubation, the cells were collected in liquid MH containing 10 mM MgCl2 (2 ml in each plate) and diluted to 0 D値〇1 (55 〇 Micron). In this 2 ml solution, 4 μl of plastid pMF121 stock solution (〇.5 μg/ml) was added and incubated at 37 ° C (with shaking) for 6 hours. The same amount of N. meningitidis B strain was used as a control group, but no plastid was added. After the incubation period, 100 microliters of the culture, or at 1/1 〇, 1/1 〇〇 and 1/1 〇〇〇 dilution, is placed at 5, 10, 20, 40 or 80. The MH plate of micrograms of erythromycin was incubated at 37 ° C for 48 hours. Colony clearing: After culturing, 2 colonies can be grown and selected from i 〇 and 2 〇 micrograms of erythromycin/ml Η ,, while there is no colony in the control group without plastid transformation. Growth. The Η44/76 wild-type strain could not grow in the selected erythromycin dish (10 to 80 μg/ml). After this day, all visible colonies were coated on a new enamel plate without erythromycin to grow. Thereafter, it is transferred to a nitrocellulose membrane (colony ablation) to examine whether or not the polysaccharide is present. In short, the colony is sucked on the nitrocellulose nanofilm and directly rinsed at -52-. The paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ---------- ----------r---book--------(Please read the notes on the back and fill out this page) 1297731 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed A7 B7 V Inventive Note (50) In PBS-0.05% Tween 2, the cells were inactivated for a further hour in PBS-0.05% Tween 20 (diluent buffer solution) at 56 °C. The membrane was then covered in a diluent buffer solution for 1 hour at room temperature (RT). Thereafter, the membrane was washed again, 3 times in a diluent buffer solution for 5 minutes, and then incubated with anti-B PS 735 Mab (Boerhinger) (diluted in 1/3 diluted in a diluent buffer solution) for 2 hours at rt. . After 5 minutes of the new washing step, the monoclonal antibody was detected by biotinylated anti-old Ig (from Aniersham) (RPN 1001), which was diluted 500-fold in the diluent buffer solution (Rt) 1 hour), proceed to the next washing step (as above). Thereafter, the membrane was incubated with a 1/1000 dilution of the streptavidin-peroxidase complex solution in the diluent buffer solution at RT for 1 hour. After the last three washing steps using the same method, the nitrocellulose membrane was used in the dark to reveal the solution (3 〇 mg of 4-chloro-1-indole solution in 10 ml of methanol plus 4 liters of PBS and 30 liters H202 37% (from Merck)) incubated for 15 minutes. The reaction is stopped in a steaming water-washing step. Whole-cell Elisas also performed whole-cell Elisas using two transformed colonies ("D,, and, R,") and wild strains (H44/76) as coating bacteria (20 μg protein/ml). And a different set of monoclonal antibodies to identify N. meningitidis. The following Mabs · anti-B PS (735 from Dr Frosch), and another Mabs from NIBSC: anti-B PS (Ref 95/750) were tested. ) anti-P1.7 (A-PorA, Ref 4025), anti-P1.16 (A-PorA, Ref 95/720), anti-Los 3,7,9 (A-LPS, Ref 4047), anti- Los 8 (L-LPS, Ref 4048), and anti-P1.2 (A-PorA Ref 95/696) 0 microtiter plate (Maxisorp, Nunc) coated with 100 μl of recombinant meningococcal B cell solution, At 37 °C (ON) and at about 20 μg/ml at -53- This paper scale applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) I.---.---- -------------^--------- (Please read the notes on the back and fill out this page) 1297731 A7 B7 V. Invention description (51 PBS. After ' Plate 3 liters of 150 mM NaCl-0.05% Tween (please read the notes on the back of the page and then fill out this page) 20 Wash three times, then over 1 〇〇 microliter of pBS-〇3% casein and incubate for 30 minutes at room temperature. The plate was washed again in the same step and then incubated with the antibody. The monoclonal antibody (1 〇〇 microliter) was different. Use at dilution factor (as shown in Figure 2) 'in PBS-0.3% peptone·〇5% Tween 20, juxtapose on a microplate' and incubate at room temperature and shake, followed by the same washing step. 1001⁄4 Ascending anti-mouse Ig (from rabbit, Dakopatts E0413) was consumed to biotin' and diluted to PBS-0.3% casein-0.05% Tween 2〇' in 1/2000 and added to the cavity to detect binding to it. Monoclonal antibody. After the washing step (as before), the plate was incubated with a chain avidin-peroxidase complex solution (100 μl Amersham RPN 1051) diluted 1/4000 in the same working solution at room temperature And 30 minutes under shaking conditions. After this incubation and final washing step, the plate was co-incubated with 1 〇〇 microliter of the original solution (4 mg of prophenyl diamine (0PD) in 10 ml of 〇·ΐ M citrate buffer solution pH 4.5, plus 5 μl of H2 〇 2) in the dark for 15 minutes. The disk is then used at 490/620 nm. Photometer read index. Results: The Ministry of Economic Affairs' Intellectual Property Office employee consumption cooperative printed Figure 1 in 20 separate colonies, which can be grown in the selection medium with erythromycin, only two ("D" and "R") Colonies showed a negative for the presence of B polysaccharide. Among others, 16 showed clear positive for B ps but were still resistant to erythromycin. This shows that it integrates the plastid into its body 'but in the wrong direction, and keeps the ps § and [ps genes intact (no double traverse). Positive and negative control groups were also tested on the plate, and it was shown that the H44/76 wild-type NmB strain was clearly positive for B polysaccharide, while meningococcal A (A1) and meningococcal c (C11) strains were resistant to _B. Ps 735 -54- Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1297731 A7 _____B7__ V. Invention Description (52)

Mab knows that it is clearly negative. These results show that about 10% of these selected colonies are correctly integrated into their genome via double transversal, while the other strain/colon is obtained from a simple cross-over, maintaining BPS and LPS gene integrity and performance. Using whole-cell Elisa, the results clearly show (Figure 2 and the table below) that the two "D &quot; and n R ''transforms (derived from D and R colonies) can no longer be confirmed by anti-B PS Mabs (735 and 95/750), anti-Los 3,7,9 and anti-Los 8 Mabs are not. However, when using the specific anti-··ΡογΑ Mabs, there is anti-P1.7 on the cells. And the clear reaction of anti-P1.16 Mabs was also observed in wild strains. No reaction was observed with non-specific anti-P〇rA Mab (anti-PI.2 mab). These results confirmed that The ProA protein and the specific P1.7 and P1.16 epitopes still exist after transformation, but the B polysaccharide and the Los 3,7,9 and Los 8 epitopes (LPS) are not. Table: Individual antibodies tested Demonstrates the direct antagonism of the tested Mabs anti-B PS 735 B polysaccharide ++ in wild strain (-) in &quot;Dn and "R" mutant anti-B PS 95/750 from NIBSC B PS ++ in wild strain ( -) in the &quot;D&quot; and ''R' mutant strains anti-Ρ1·7 (NIBSC) Porin A loop 1 ++ in all wild strains and mutant strains anti-P1.16 (NIBSC) Porin A ring Take 4 ++ in all wild strains and Mutant anti -Los 3,7,9 LPS ++ on the wild type strain (i) '' D "and" R "mutants -55-

1 The paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm T - 1 II, -------· I--l·---^« — 1 —----- (Please read the note on the back? Please fill out this page again) Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1297731 A7 ____B7 V. Invention Description (53) Anti-Los 8 (NIBSC) LPS +/- in wild strain ( -) in the &quot;D" and mutant anti-P1.2 (NIBSC) anti-Porin A blood subtype 1.2 (-) in all wild type and mutant strains in the case of 2 · construct a diverse gene delivery vector (PCMK The series) allows for the homologous recombination and the stable integration of foreign DNA into the porA region of N. meningitidis by integration with the p〇rA region of the N. meningitidis constituting the plastid. (gene, operon and/or sputum expression) can be used to construct a strain of N. meningitidis that produces a recombinant blister with improved recombination. Typically, this vector contains: (1) the plastid skeleton can be in the large intestine Replica in bacilli, but N. meningitidis (suicide plastid) '(2) at least one, but better two homogenous regions, making the whole Can be aligned to chromosomal regions, such as porA, (3) functionally efficient greening (promoter, regulatory region and terminator) and translation (the most appropriate ribosome binding site) in N. meningitidis And start codon) signals, (4) multiple colonization sites and (5) genes for screening, allowing the plastid to remain in E. c〇li and the selection of integrons in N. meningitidis. Additional requirements include, for example, absorption sequences to facilitate foreign DNA entry into N. meningitidis, and conversely screenable markers such as sacB, rpsL, gltS to enhance the frequency of double transverse events. Named pCMK, which is shown in Figure 3. Its equivalent complete nucleotide sequence is shown in SEQ ID NO: 1. pCMK is derived from the pSL1180 backbone (Pharmacia Biotech, Sweeden), a copy that can be replicated in Ε· coli High number of plastids with bla gene (and therefore -56- This paper scale applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm) -----.-------- ------r---book--------- (please read the notes on the back and fill out this 1297731 A7 _____________B7 V. Description of the invention (54) for resistance to ampicillin). In addition, pCMK functionally contains two porA contiguous regions necessary for homologous recombination (porA5, and p〇rA3, containing a transcriptional terminator) Providing a selectable marker for resistance to oxytocin, two absorbing sequences, the porAAacO chimeric promoter is inhibited in the E. c〇li host expressing iaciq but transcription in N. meningitidis Active, and the multiple selection sites necessary for the insertion of foreign DNA into pCMK (there are 5 positions: Ndel, Kpnl, Nhel, PinAl, and SphI) 〇pCMK is constructed as follows, porA5' and 卩〇^3' have recombination regions, The porA/lacO promoter was amplified by PCR amplification of the oligonucleotides listed in the table below, and was sequenced in pTOPO and sequenced. These DNA fragments were sequentially excised from pTOPO and colonized into PSL1180. The amphotericin-resistant sputum was excised from pUC4K (Pharmacia Biotech, Sweeden) and introduced between the porA5' contiguous region and the porA/lacO promoter region. (Please read the note on the back and then fill out this page.) Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed -57- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) Ministry of Economic Affairs Intellectual Property Bureau Employee Consumption Cooperative Printed 1229731 A7 B7 V. Description of Invention (55) Table: Oligonucleotide oligonucleotide citrate for this work 1 PorA5' Fwd y-CCC AAG CTT GCC GTC TGA ATA CAT CCC GTCATTCCTCA-3, Mmilll selection site absorption sequence (J PorA5'Rev 5, CGA TGC TCG CGA CTC CAG AGA CCT CGT GCG GGC C-3' colonization position PorA3' Fwd y-GGA AGA TCr GAT TAA ATA GGC GAA AAT ACC AGC TAC GA-3* Colonization Site Stop Codon (J PorA3, Rev 5f-GCC GAA TTC 7TC AGA CGG C GC AGC AGG AAT TTATCG G-3f Colonization Site Absorption Sequence (J PoLa Rev 1 y· GAA TTG TTA TCG GCT CAC AAT TCC GGG CAA AC A CCC GAT AC-3 ' PoLa Rcv2 5*-GAA TTC CAT ATG ATC GGC TTC CTT TTG TAA ATT TGA TAA AAA CCT AAA AAC ATC GAA TTG TTA TCC GCT C-3* Selection Site PorAIacO Fwd 5,-AAG CTC TGC AGG AGG TCT GCG CTT GAA TTG^1 m selection PorAlacO Rev 5'-CTT AAG GCA TAT GGG CTT CCT TTT GTA A-3, MW colonization position PPA1 5, · GCG GCC GH GCC GAT GTC AGC C-3, PPA2 5,-GGC ATA GCT GAT GCG TGG AAC TGC- 3' N-full-Ol: 5'-GGG AAT TCC ATA TGA AAA AAG CAC TTG CCA CAC-3' Ndel colonization position Nde-NspA-3: 5,- GGA ATT CCA TAT GTC AGA ATT TGA CGC GCAC-3 'Ndel colonization position PNS1 5'- CCG CGA ATT CGG AAC CGA AC A CGC CGT TCG-3, £〇7RI colonization position PNSl 5, · CGT CTA GAC GTA GCG GTA TCC GGC TGC -3' along the αΙ selection site PromD15-51X 5 - GGG CGA ATT CGC GGC CGC CGT CAA CGG CAC ACCCGTTG-3' EcoRI and Notl colonization position PromD15-S2 5*- GCT CTA GAG CGG AAT GCG GTT TCA GAC G-3, colonization position PNS4 5' - AGC TTT ATT TAA ATC CTT AAT TAA CGC GTC CGG AAA ATA TGC TTA TC J4 Swal and PacI selection position PNS5 51- AGC TTT GTT TAA ACC CTG TTC CGC TGC TTCGGC-3' Pmel selection position D15-S4 7 5, · GTC CGC ATT TAA ATC CTT AAT TAA GCA GCC GGA CAG GGC GTG G-3' Swal and PacI selection position D15-S5 5^- AGC TTT GTT TAA AGG ATC AGG GTG TGG TCG GGC-3* Pmel Position -58- (Please read the note on the back and fill out this page.) This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm). 1297731 A7 B7 Ministry of Economic Affairs, Intellectual Property Office, employee consumption cooperative, Description of the invention (56): Example 3: The regulation of the antigenic content of the potential antigen PorA'-outer membrane large blister is beneficial in improving its safety and efficacy for use in vaccines, or for diagnostic or therapeutic use. Components such as N. meningitidis serum B group capsular polysaccharide should be removed to rule out the risk of autoimmune immunity (see Example 1). Similarly, the immunological advantage of the main membrane antigen is suppressed, such as PorA, which is beneficial, can produce strain-specific bacteriostatic antibodies, but does not provide cross-protection. To illustrate this approach, we use the PCKK(+) vector to construct Neisseria meningitidis serum B group bacteria. It lacks capsular polysaccharide and immunogenic p〇rA outer membrane protein antigen. For this purpose, the deletion of porA gene was introduced into H44/76, cps-strain by homologous recombination as described in Example H. The /76 cps-strain was prepared to be competent and transformed with 2 micrograms of supercoiled pCMK(+) plastid DNA as previously described. A portion of the transformed mixture (100 microliters) was plated onto the Mueller-Hinton plate. On the above, add kunmycin (200 μg / ml), and then incubated at 37 ° (: 24-48 hours. Select the colony resistant to Kang Huisu) and then apply it on ΜΉ-Κη, and Incubate for another 24 hours at 37 ° C. At this stage, half of the bacterial culture is used to make Glycerol stock solution (15% v/v), then chilled at -70 ° C. Another (about 1 〇 8 bacteria) was resuspended in 15 μl of distilled water, boiled for 1 〇 minutes, used for PCR screening The template is used to synthesize two internal porA primers, called PPA1 and PPA2, and used for the upstream PCR amplification of the boiled bacterial lysate, as described by the supplier (HiFi DNA polymerase, Boehringer Mannheim, GmbH) The thermal cycle used is as follows: 2 5 times (94 ° C, 1 minute, 52 ° (:, 1 minute, -59- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) I, - ----..----^--------- (Please read the notes on the back and fill out this page) 1297731 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperatives Print A7 B7 V. Invention Description (57) 72 ° C, 3 minutes) and 1 time (72 ° C, 10 minutes, 4 ° C until recovery). Due to the double traversal effect between the pCMK DNA chromosome porA region will be deleted # 1 and #2 back The region necessary for cold, so the pure line lacking the U70 bp PCR amplified fragment was selected as the porA deletion mutant. These pcR results can be analyzed in parallel, at considerable The presence of porA in the bacterial protein extract was further confirmed. For this purpose, another bacterium (estimated to be 5 · 1 〇 8 bacteria) was resuspended in 50 μl of PAGE_SDS buffer solution (SDS 5%, glycerol) 30% ' 0-3⁄43⁄4 base ethanol 15% 'bromine blue, 〇 · 3 mg / ml, Tris-HCl 250 mM pH 6.8), boiled (l 〇〇 ° C) frozen (-20 Ό) / boiled (1 〇 Hey. 〇 three times' and electrophoresed on a 12.5% gel by PAGE-SDS. The gel was then stained with Coomass Brilliant R250 or transferred to a nitrocellulose membrane and probed with anti-_1 &gt; 〇1^ monoclonal antibodies as described by Maniatis et al. As shown in Figure 4, both Coomassie blue and immunostaining staining confirmed that the p〇rA PCR-negative pure line did not produce measurable and comparable levels of P〇rA. This result confirmed that the pCMK vector was functional and successfully used to align DNA into the porA gene while eliminating the production of PorA outer membrane protein antigen. Example 4: Slotial regulation of NspA outer membrane protein produced in large water cancer, the disc derived from the porA &amp; cps gene-deficient recombinant meningococcal Neisseria blood slag B 薜嫉 has protective antigen Jiafeng's large vesicular sac can be beneficially used to improve the efficacy and scope of the outer membrane protein-based vaccine. In this case, the recombinant N. meningitidis strain lacking the functional CpS and p〇rA genes will be genetically manipulated, so that the expression level of the outer membrane protein NspA can be positively controlled. For this purpose, the gene that directs the synthesis of NspA utilizes the NO 1 - -60-, -, and the tensor scale to the Chinese National Standard (CNS) A4 specification (210 X 297 mm). J,------- ----^----订-----1 (Please read the note on the back and then fill out this page) Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1297731 A7 ____B7_ V. Invention description (58) Complete The -Ndel and Ndell oligonucleotide primers were subjected to PCR amplification (see Table 2). The conditions for PCR amplification are as described by the supplier (HiFi DNA polymerase, Boehringer Mannheim, GmbH). The thermal cycle is as follows: · 25 times (94 ° C, 1 minute, 52 ° C, 1 minute, 72 °) C, 3 minutes) and 1 time (72 ° C, 10 minutes, 4 ° C until recovery). A comparable expander was hydrolyzed with Ndel and re-inserted into the Ndel restriction site of the pCMK(+) delivery vector. Check the direction of embedding, and the recombinant plastid was named pCMK(+)-NspA. It was purified on a large scale by QIAGEN's largest preparation kit. Two micrograms of this material was used to transform the meningitis of Neisseria lacking the functional cps gene. Coccurrence serum B-type strain (strain as described in Example 1). Using the combination of PCR and the Western blotting step of Example 3, the integration effect caused by the double traversal effect between the pCMK(+)-NspA vector and the porA region of the chromosome was selected. The bacteria (equivalent to about 5.108 bacteria) were resuspended in 50 μl of PAGE-SDS buffer solution, frozen (-20 ° C) / boiled (l ° ° C) three times, and then electrophoresed by PAGE-SDS Isolation was performed on a 12.5% gel. The gel was stained with Coomassie Brilliant Blue R250 or transferred to a nitrocellulose membrane and probed with anti-NspA multi-strain serum. Both Coomassie Brilliant Blue (data not shown) and immunostaining staining (see Figure 4) confirmed that porA PCR-negative pure lines did not produce detectable and comparable levels of porA. The performance of NspA is examined in whole cell bacterial lysate products (WCBL) or in membrane large water cancer preparations derived from NmB [cps-, porA-] or NmB [cps-, porA-, Nspa+]. Although no differences were observed by Coomassie staining, immunosuppression with anti-NspA multiple strains of serum, in the performance of NspA (in terms of endogenous NspA levels), WCBL and adventitial large blister preparations (see Figure 5) Both are measured and increased by 3-5 times. This result confirms that 1) 〇^{:(+)-valent 0-person carrier is functional-61 - This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) ----'-- ----------l·---订--------- (Please read the phonetic on the back? Please fill out this page again) 1297731 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing A7 ___ B7_ V. Inventive Note (59), and can be successfully applied to the outer membrane protein, such as NspA, which is positively regulated and at the same time eliminates the production of PorA outer membrane protein antigen. Example 5: D15/Omp85 膜 membrane protein antigen is positively regulated in large water cancer, its name is axillary from lack of function {sheng cps gene but can express the weight of PorA, group N. meningitidis serum B Some geographically isolated populations (such as Cuba) are infected by a limited number of N. meningitidis isolates, most of which belong to one or less outer membrane protein serotypes. Since PorA is the major outer membrane protein antigen that protects against strain-specific bacteriostatic antibodies, the use of a limited number of porA serotypes in vaccines will likely provide vaccine protection. In this condition, it would be beneficial for PorA to be present in the outer membrane vesicle, and enhancing the vaccine efficacy of this recombinant can improve the large leeches. However, this vaccine containing PorA can be further improved by increasing the level of other cross-reactive OMPs, such as omp85/D15. In the following examples, the pCMK(+) vector was used to positively regulate the expression of the Omp85/D15 outer membrane protein antigen in strains lacking a functional cps gene but expressing porA. For this purpose, the gene for the synthesis of Omp85/D15 was used to perform PCR amplification using the D15-Ndel and D15-NotI oligonucleotide citrate primers. The conditions for PCR amplification are as described by the supplier (HiFi DNA Polymerase, Boehringer Mannheim, GmbH). The thermal cycle was as follows: 25 times (94 ° C, 1 minute, 52 ° C, 1 minute, 72 ° C, 3 minutes) and 1 time (72 ° C, 10 minutes, 4 ° C until recovery). A considerable expander is embedded in the pTOPO selection vector according to the instructions of the operation, and then confirmed by the sequence. This Omp85/D15 DNA fragment was excised from pTOPO by Ndel/Nsil restriction hydrolysis and colonized to a comparable restriction site in the pCMK(+) delivery vector. Recombinant plastid, named -62- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) ----.---------------- -------- (Please read the phonetic on the back? Please fill out this page again) 1297731 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 _____B7_________ V. Invention description (60) for pCMK(+)-D15, The QIAGEN mass production kit was extensively purified, and 2 μg of this material was used to transform the N. meningitidis serogroup B strain (strain described in Example 1) lacking the functional cps gene. To preserve the performance of p〇rA, a combination of PCR and Western blotting steps was performed to select for integration by a single cross (either in Omp85/D15 or P〇rA). The Kangmycin resistance was positive by porA·specific PCR and western blot test, and stored in glycerol stock solution at -70 °C, and then used in further research. Bacteria (corresponding to approximately 5.2 〇8 bacteria) were resuspended in 50 μl of PAGE-SDS buffer solution, frozen (-20 ° C) / boiled (1 〇〇. 〇 three times, then pAGE_ SDS electrophoresis Isolation on a 12.5% gel. The gel was stained with Coomassie Brilliant Blue R250 or transferred to a nitrocellulose membrane and probed with anti-p〇rA monoclonal antibody. As shown in Figure 6, Coomassie blue and immunosuppression Staining confirmed that P〇rA Pcr-positive pure line could produce PorA. The performance of D15 was examined using a large blister preparation derived from NmB [cps-, porA-] or NmB (cps-, p〇rA+, D15+). Smear can detect a significant increase in performance levels on the D15 formulation (in terms of endogenous D15 levels) (see Figure 6). This result confirms that the pCMK(+)_D15 variant is functional and can be used successfully. To positively regulate the performance of the outer membrane protein, such as D 〖5, without eliminating the production of the major PorA outer membrane protein antigen. Example 6: i-like promoter delivery tray structure filling. The reason for this method is shown in Figure 7, and can be integrated into 7 necessary steps. Some of these steps are described below, the carrier is constructed to positively regulate NspA and D15/O. Performance of mp85. Positive regulation of NspA gene expression -63- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm)

Tilt-------βι--------^---------Αν. (Please read the notes on the back and fill out this page) __ Ministry of Economic Affairs Intellectual Property Office staff consumption Co-operatives Printed 1229731 A7 _____ V. INSTRUCTIONS (61) Step 1 • The DNA region upstream of the NspA-encoding gene (997 bp) (SEQ. ID N0:2) can be found in the private Incyte PathoSeq database, which contains meningitis Neisser. The genotype DNA sequence of the untreated strain of the bacterium strain ATCC 13090. Using this sequence, two oligos called PNS1 and PNS2 can be designed: y: acid primer (see Table 2) and synthesized. These primers can be used for PCR amplification, in which genomic DNA extracted from the H44/76 strain is utilized. Step 2. A similar expander was cleared using the Wizard PCR kit (Promega, USA) and subjected to hydrolysis of EcoRI/Xbal restriction enzyme for 24 hours, as described by the manufacturer (Boehringer Mannheim, Germany). The equivalent DNA fragment was gel purified and then placed in the equivalent position of the pUC 18 selection vector. Step 3 • Recombinant plastids Prepare on a large scale and take one more template for reverse PCR amplification. Reverse PCR using PNS4 and PNS5 oligonuclear acid: acid, the thermal cycling conditions utilized are as follows: 25 times (94 ° C, 1 minute, 50 ° C, 1 minute, 72 ° C, 3 minutes) and 1 time (72 ° C, 1 〇 min, 4 ° C until recovery). A linearized pUC 18 vector is obtained in which there is a deletion in the intervening region of the upstream region of NspA. Steps for positive regulation of D15/omp85 gene expression 1 • DNA region (1 〇〇〇 bp) (SEQ. ID NO: 3) that falls upstream of the weeping coding gene can be found in the private inCyte path〇Seq database. , which contains the untreated DNA sequence of Neisseria meningitidis ATCC 13090. Using this sequence, two oligo nuclei called PromD15_51x and Pr 〇mD15-S2 (see Table 2) can be designed and synthesized. These primers can be used for PCR amplification using genomic DNA extracted from the H44/76 strain. Step 2 • The equivalent expander was removed using the wizard PCR kit (Promega, USA) and then subjected to the hydrolysis of Ec〇RI/xbaI restriction enzymes. -64. This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297). Public love) ----------------r----book--------- (please read the notes on the back and fill out this page) 1297731 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 ___B7_ V. Invention description (62) 2 4 hours, conditions as described by the manufacturer (Boehringer Mannheim, Germany). A comparable DNA fragment was gel purified and embedded in a comparable position in the pUC 18 selection vector. Step 3. The recombinant plasmid was prepared on a large scale, and a template was prepared for the amplification of the reverse PCR. Reverse PCR was performed using D15-S4 and D15-S5 oligonucleotides using the following thermal cycling conditions: 25 times (94 ° C, 1 minute, 50 ° C, 1 minute, 72 ° C, 3 minutes) And once (72 ° C, 10 minutes, 4 ° C until recycling). A linearized pUC 18 vector with deletion in the upstream region of D15/omp85. Example 7: Fermentation process for producing recombinant large blisters The following example describes a method for producing recombinant large blisters, in which pods are absent Membrane polysaccharide or capsular polysaccharide and PorA. This step can be applied to a wide range of Neisseria meningitidis recombinant strains and can be adapted to an expanded scale. Medium: Neisseria meningitidis serogroup B strains were propagated on solid phase (FNE 004 AA, FNE 010 AA) or liquid phase (FNE 008 AA) medium. These new culture media for culture of meningococcus are beneficially free of animal products and can be considered as further aspects of the invention. Component FNE 004 AA FNE 008 AA FNE 010 AA Agar 18 g / l - 18 g / l NaCl 6 g / l 6 g / l 6 g / l glutamine sodium 1.52 g / l a NaH2P04.2H20 2.2 g / 2.2 g / l 2.2 g / l KC1 0.09 g / l 0.09 g / l 0.09 g / l NH4C1 1.25 g / l 1.25 g / l 1.25 g / l glucose 5 g / l 20 g / l 5 g / l -65 - This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) I---.------------r---book-------- - # (Please read the notes on the back and fill out this page) 1297731 A7 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed V. Inventions (63)

1 : This method is carried out in two steps, including pre-culture on a solid phase medium, followed by liquid phase culture. Solid phase pre-culture······································································································· . The pasteurian dish is at 37. Underarm cultivation for 18 ± 2 hours. The surface growth was resuspended in 8 ml of FNE008AA (see above) with 15 g/L of erythromycin added. The flask was incubated. 2 ml of the resuspended solid phase was preincubated and added to 2 liters of a flask containing 400 ml of FNE008AA, to which 15 g/l of erythromycin was added. The flask was placed on a shaker plate (2 rpm) and incubated at 37 C for 16 ± 2 hours. The cells were centrifuged at 5000 g for 15 minutes from the culture broth and separated at 4 °C. II. Neisseria meningitidis serum type B cps-recombinant large water brigade batch ridge _: This is carried out in three false steps, including pre-culture, liquid phase culture and batch mode culture on solid phase medium. The solid phase pre-culture, the vial of the strain was removed from the cold bundle chamber (-80 C), and the solution was decanted to room temperature, and then ι·1 ml was applied to the pasteurized blood containing 15 ml of FNE004AA (see above). The barley dish was incubated at 37 ° C for 18 ± 2 hours. The surface growth was resuspended in 8 ml of FNE008AA supplemented with 15 mg/L erythromycin (see above). Liquid phase pre-culture. 2 ml resuspension -66 This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) ----.-----------------^-- -------- (Please read the notes on the back and fill out this page)

1297731 A7 V. INSTRUCTIONS (64) A floating solid phase preculture is added to a 2 liter flask containing 400 ml of FNE® 8AA with 15 mg/liter of erythromycin added. The flask was shaken on a plate (200 rpm) and incubated at 37 ° C for 16 ± 2 hours. The contents of the flask can be used to inoculate 20 liters of acid fermentation tank. The batch mode is cultured in a fermentation tank. The inoculum (400 ml) was added to a pre-sterilized 2 liter (total volume) fermentation tank containing 1 liter of FNE008AA supplemented with 丨5 mg/liter erythromycin. ? 11 调整 Adjust and maintain at 7.0 with NaOH (25% w/v) &amp; H3P04 (25% v/v). The temperature is regulated at 37C. The aeration rate was maintained at 2 liters of air per minute and the dissolved oxygen concentration was maintained at 20% saturation by controlling the agitation rate. The residual pressure in the fermentation tank was maintained at 300 g/cm 2. 9 ± 1 hour and the culture was in a steady state. The cells were centrifuged at 5000 g at 4 ° C and separated from the culture broth for 15 minutes. Neisseria meningitidis serum B group cps-, PorA-recombinant large water disease burning turtle culture • This is carried out in two steps, including pre-culture in solid phase medium followed by liquid phase culture. Solid phase pre-culture. The vial of the strain was self-cold; the east chamber (_8〇. removed from the sputum, thawed to room temperature, and then 0.1 ml was applied to the pasteurized blood containing 15 ml of FNE010AA (see above). The pasteurized dish was at 37 °C. The soil was incubated for 18 hours for 2 hours. The surface growth material was resuspended in 8 ml of FNE008AA (see above), and 200 mg/liter of oxytetracycline was added thereto. The flask was cultured. 2 ml of the resuspended solid phase preculture to 2 The flask containing 400 ml of FNE008AA was added with 200 mg/liter of oxytetracycline. The flask was placed on a shaking plate (200 rpm) and incubated at 37 ° C for 16 hours for 2 hours. The cells were incubated with broth at 5000 g. Isolation was carried out by centrifugation at 4 ° C for 15 minutes. Example 8: Isolation and purification of large blisters from meningitis of capsular polysaccharide from the capsular polysaccharide. The recombinant large blisters were purified as follows. The cell paste (42 g) was suspended at 211 m. -67- This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) 1—IΙΓ — — — — — -----r------I-- (Please read first On the back of the phonetic? Matters fill out this page) Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1297731 Ministry of Economic Affairs Intellectual Property Bureau Consumer Cooperatives Printed A7 ________B7____ V. Inventive Note (65) 0.1 M Tris-Cl buffer solution, pH 8.6, containing 10 mM EDTA and 0.5% sodium deoxycholate (DOC). The ratio of buffer solution to bioclusters is 5/1 (V/W). The bio-branches were extracted by magnetic stirring for 3 minutes at room temperature. The total extract was further centrifuged at 20,000 g for 30 minutes at 4 ° C (in the JA-20 rotor). 13,000 rpm, Beckman J2-HS centrifuge). Discard the pellet. The supernatant was centrifuged at 125,000 g for 2 hours at 4 °C (40,000 rpm on a 50.2 Ti rotor, Beckman L8-70M ultracentrifuge) to concentrate small The supernatant was discarded. The pellet was gently suspended in 25 ml of 50 mM Tris-Cl buffer solution, pH 8.6, containing 2 mM EDTA, 1.2% DOC and 20% sucrose. Over 125,000 g at 4 C over centrifugation 2 After the second centrifugation of the hour, the capsule was suspended in 4 4 ml of 3% sucrose and stored at 4 ° C. All solutions for the extraction and purification of large blisters contained 〇·〇1% thimerosal. As described in Figure 8, this method produces protein preparations that are highly enriched with outer membrane proteins, such as PorA and PorB. , Example 9: Identification of a bacterial promoter suitable for the forward regulation of antigen-encoding genes In order to obtain a positive regulation of the gene for the synthesis of the outer membrane protein, it is necessary to use a strong bacterial promoter element. Under this circumstance, we have previously shown that the positive regulation of N. meningitidis nSpA, hsf and 〇mp85 gene by the PorA promoter allows us to isolate equivalent NspA, Hsf and 〇mp85 proteins. Feng's recombinant large blisters. In addition to p〇rA, promoters can be used to achieve different levels of positive regulation to overcome possible p〇rA phase changes and/or to achieve adaptive gene expression (iron-regulated promoters). Here I describe the method to identify the precise green start position of a strong promoter element that appears to provide a high level of performance (in bacteria). Due to the promoter regulation requirements, the pass-68-^ paper scale is suitable _ _ standard (CNS) A4 specification (21Q X 297 i¥) :----------^--------tr --------- (Please read the note on the back and then fill out this page) 1297731 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 —__B7_ V. Invention description (66 ) often included in the distance + 1 position Up to 200 bp upstream and 50 pb downstream ((: 〇11&amp; (1〇-Vides J, Magasanik B, Gralla JD, 1991, Microbiol Rev 55(3): 371-94), the results of this experiment made us identifiable Approximately 250 bp of DN A fragment with strong promoter activity. Major outer membrane proteins such as Neisseria meningitidis PorA, PorB &amp; Rmp, H. influenzae PI, P2, P5 & P6, catarrh OmpCD, OmpE, and certain lipids and/or iron-regulated proteins of these bacteria have strong promoter requirements. Under the general method of confirmation, we have utilized the rapid expansion of cDNA elements (5, RACE). The transcription initiation sites of the strong N. meningitidis PorA and PorB promoters were located. The principle of 5' RACE is as follows: 1) Using QIAGEN &quot;RNeasy&quot; Total RNA was extracted group Genomic DNA DNase treatment to remove, re QIAGEN purification;. 2) mRNA using a porA specific 3 'end primers (referred porA3) recorded row inversion effect. The expected size of the CDNA6 was 307 nt. RNA was removed by alkaline hydrolysis; 3) The single strand DNA oligo was immobilized (i.e., DT88) to the 3' end of the cDNA using T4 RNA ligase. Expected product size: 335 nt. Expanding and immobilizing the cDNA using a semi-stable PCR combination; 4) PCR amplification with a fixed-stranded cDNA, in which the primer is fixed with a complementary sequence as a 5' primer (ie DT89) and 3, a primer (ie pl-2), which is inside the 3rd end RT primer PorA3. Expected product size: 292 bp; 5) PCR amplification of previous PCR products, using 1&gt; Ding 89 for 5|end primer and ? 1-1 is the 3' end primer (inside 1-2). The expected product size; 211 bp; and 6) sequenced with the pl-1 primer (expected product size can be estimated since the PorA transcription start position is known: 59 nt before the nATGn translation start position. -69- This paper scale applies to China National Standard (CNS) A4 specification (21〇X 297 mm) ----r-------------- (Please read the notes on the back and fill in the form) Page) Order --- · 1297731 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 _ B7 __ V. Description of invention (67) Experimental procedure Total RNA from about 109 cells of Neisseria meningitidis serum B group cps- Extraction of porA+ strain. Extraction of 1 ml of liquid culture at appropriate optical density (〇D6QQ=l) according to the manufacturer's instructions in QIAGEN &quot;RNAeasy' kit. Removal of chromosomal DNA is to add 10 units of DNase without RNase (Roche Diagnostics, Mannheim, Germany) to 30 μl of eluted RNA and incubated for 15 min at 37 ° C. DNA-free RNA was purified using the same QIAGEN kit according to the manufacturer's instructions. Using primers porA3 and 200 The unit's SUPERSCRIPT II reverse transcriptase (Life Technologies) performs reverse transcription reactions. Should be performed in a volume of 50 μl containing: 5 μl of 2 mM dNTP, 20 picomol of p〇rA3 primer, 5 μl of 10X Superscript II buffer drop 9 μL of 25 mM MgCl2, 4 μg L0·1Μ DTT, 40 units of recombinant ribozyme inhibitor and 1 microgram of total RNA. The porA3 primer is gradually cooled back (70°C, 2 minutes, 65°C, 1 minute, 60°C, 1 minute, 55 °C, 1 minute, 50 ° C, 1 minute and 45 ° C 1 minute) 'Add Superscript II. RT reaction was carried out at 42 C for 30 minutes, then 5 cycles (1 minute at 50 ° C, 53 ° C, 1 minute and 56 ° C 1 minute knock) to destabilize the RN A - level structure. Two sets of parallel reactions were carried out, and the green enzyme was reversed in the negative control reaction. RNA was hydrolyzed by hydrolysis. Remove and add 1 μl of 0.5 μ EDTA followed by 12.5 μl of 0·2 M NaOH and incubate for 5 minutes at 68 ° C. Add 12.5 μl of 1 M Tris-HCl (pH 7.4). And, add 20 micrograms of glycogen (R 〇che ]V1 ο 1 ecu 1 ar B i 〇che in ica 1 s,]V1 a η nheim,G er Hi any), 5 μl of 3 M sodium acetate and 6 0 microliters of isopropanol . Both samples are resuspended -70-^paper scale applicable to China National Standard (CNS) A4 specification (210 X 297 mm) ----.------------ (Please read the back Note: Please fill out this page) Order--- # 1297731 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Print A7 —_______B7 _ V. Invention Description (68) in 20 μl of 1〇: 1 TE (10 mM Tris- HCl, pH 7.4; 1 mM EDTA, pH 8). T4 RNA ligase was used to immobilize 5,-phosphonium, and 3, terminal ddCTP-blocked immobilized virulence acid DT88 (see table below). Two parallel ligation reactions were performed at room temperature overnight, each containing: 1.3 μl of ι〇χ rnA ligase buffer solution (Roche Molecular Biochemicals), 0.4 &quot; M DT88, 10 μl of cDNΑ or RT control sample, And 3 units of Τ4 RNΑ ligase. As for the negative control group, a second set of ligation reactions was performed, and T4 RNA ligase was omitted. The resulting ligation reaction mixture can be used directly without further purification in the next PCR. The immobilized ligation cDNA was amplified using a semi-hidden and heat-activated PCR combination to increase specificity and product yield. Four separate first rounds of PCR were performed in the RT/ligase reaction and the control group, containing 3 μl of each of the 3 μl microliters of volume 〇χ Taqming buffer solution '3 μL 25 mM MgCl2, 1 μg Increasing 10 mM dNTPs, each 1 〇 pico-mole primer and i microliter equivalent RNA ligation reaction. The PCR was thermally initiated using Taq Pin (Life Technologies) DNA polymerase (2 units added). The first ligation-fixed PCR (LA-PCR) was performed, and a specific primer DT89 and a transcript-specific primer pl_2 (see table below) were immobilized using 1 〇 micromolar, which was introduced at the 3' end RT primer porA3. Nei Shao. The PCR was performed with 95 χ: 5 minute steps (for DNA polymerase activation)' followed by 1 cycle of 95 ° C, 10 seconds and 70 ° C for 1 minute (1 cycle per cycle). 15 cycles of 95 ° C, 1 〇 and 6 〇Ό 1 minute. Perform a second round of semi-hidden LA-PCR under the same conditions, using primers DT89 and pl-2 internal primers, plus 丨〇 micro-mole (see table below) and _____ -71 - this paper size applies China National Standard (CNS) A4 Specification (210 X 297 mm) ____.----------------- Order--------- (Please read the back Note: Please fill out this page again) Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1297731 A7 _____^_ B7_ V. Invention Description (69) 1 microliter of the first round of PCR. The expanded product was purified using QIAGEN &quot;OIAquick PCRJ purification&quot; kit H-instruction before being sequenced. The RACE PCR product was sequenced using a CEQTM dye terminator cycle sequencing kit (Beckman, France) using a 10 picomol primer pl_1. The sequencing reactions were performed according to the instructions provided and the sequencing products were analyzed by the Ceq2000 DNA Analysis System (Beckman-Coulter). DT88 5f GAAGAGAAGGTGGAAATGGCGTTTTGGC 3f DT89 5f CCAAAACGCCATTTCCACCTTCTCTTC 3f porA3 5' CCAAATCCTCGCTCCCCTTAAAGCC 3, p 1 -2 5 CGCTGATTTTCGTCCTGATGCGGC 3f p 1 -1 5* GGTCAATTGCGCCTGGATGTTCCTG 3, N. meningitidis porA promoter results using the 5'-RACE step previously described The transcription start point of the porA-mRNA of the N-type starter code 59 bp upstream and the N. meningitidis serotype B (H44/76 strain) porA-mRNA was determined. This result confirms the mapping made by the extension of the primer and was developed by van der Ende et al (1995). This result supports: For porAATG, DNA fragments containing -9 to -259 are suitable for driving strong genes in N. meningitidis and are also possible in other bacterial species, such as Haemophilus, Mora Pseudomonas, Pseudomonas. Results of the N. meningitidis porB promoter The same experimental strategy can also be applied to the mapping of the transcript origin of porB of the N. meningitidis serogroup B group (H44/76 strain). The primers listed in the table below correspond to the 3*-terminal RT primer (porB3), a trans-green-specific primer, which is internal to p0rB3 (porB2) and porB2 (porB 1). p〇rB3, porB2 and porB 1 were separated by 265 b, 195 bp and 150 bp downstream of the ATG start codon. -72- This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) I.---.------------- l·------- ----- (Please read the notes on the back and fill out this page) 1297731 A7 B7

5. Description of the invention (7G porB 1 5, GGTAGCGGTTGTAACTTCAGTAACTT 3, porB2 5f GTCTTCTTGGCCTTTGAAGCCGATT 3f porB3 5, GGAGTCAGTACCGGCGATAGATGCT 3, using the porBl and DT89 primers, a PCR expander of ~200 bp can be obtained by performing 5'-RACE mapping. porBl is located at 150 bp of the porB ATG start codon. This result supports the following: porB transcription initiation position is about 50 bp (+/- 30 bp) upstream of porB ATG. The acid, currently determined by DNA sequencing, supports the following results, ie, for the porB ATG start codon, a DNA fragment containing -1 to -250 is suitable for driving a strong gene in N. meningitidis Performance, and other fungi, such as Haemophilus, Moraxella, Pseudomonas may also be. Example 10: Proactive replacement of the positive regulator of Neisseria meningitidis serum B type, please read the back of the note Item I I page IIIIIII Department of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing

The Omp85 gene was designed to replace the endogenous promoter region of the D15/Omp85 gene with a strong porA promoter to positively regulate the production of D15/Omp85 antigen. For this purpose, the promoter was substituted for the plastid by the E. coli selection method. The DNA region (1000 bp) upstream of the D15/Omp85-encoding gene can be found in (SEQ ID NO: 3) a private Incyte PathoSeq database containing the unprocessed genomic DNA sequence of the N. meningitidis ATCC 13090 strain. The main steps of this step are shown in Figure 9. Briefly, for the D15/Omp85 gene start codon (ATG), the DNA fragment covering -48 to -983 (1000 bp) utilizes the oligonucleotide PorD15 containing the EcoRI and Xbal restriction positions (underlined), respectively. -51X (5f-GGG CGA ATT CGC GGC CGC CGT CAA -73- This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm) A7 1297731 ______Β7___ V. Description of invention (71) CGG CAC ACC GTT G -3,) and PorD15-52 (5,-GCT CTA GAG CGG AAT GCG GTT TCA GAC G-3,) were subjected to PCR amplification. This fragment is subjected to limited hydrolysis and is then embedded in the pUC18 plastid bound by the same enzyme. The resulting construct was subjected to in vitro mutagenesis using a Genome Priming system (using pGPS2 donor plastids) from New England Biolabs (MA, USA). A pure line (derived from Tn7 with a chlorophyll resistance gene) embedded with a mini-trans factor was selected. The pure line containing the mini-trans factor embedded therein was isolated in the D15/Omp85 5* contiguous region, 401 bp downstream from the EcoRI position, and was used for further studies. This plastid undergoes cyclic PCR mutagenesis (Jones &amp; Winistofer (1992), Biotechniques 12: 528-534) to (i) delete the repetitive DNA sequence (Tn7R) produced by translocation, (ii) The meningococcal absorption sequence necessary for insertion into the transformation, and (iii) embedding in a suitable restriction position for exogenous DNA, such as a promoter, for colonization. Cyclic PCR was performed using TnRD15-KpnI/XbaI + US (5,-CGC CGG TAC CTC TAG AGC CGT CTG AAC CAC TCG TGG ACA ACC &lt;:-3?) and TnR03Cam (KpnI) (5,-CGC CGG) TAC CGC CGC TAA CTA TAA CGG TC-3') Oligonucleotide, which contains suitable sequences for the absorption sequence and underlined (ΚρηΙ and Xbal). The resulting PCR fragment was gel purified, hydrolyzed with Asp718 (ΚρηΙ isozyme), ligated into the 184 bp DNA fragment containing the porA promoter, and then used PorA-01 (5?-CGC CGG CGA GGT CTG CGC TTG AAT) TGT G-3,) and PorA02 (5'-CGC CGG TAC CTC TAG ACA TCG GGC AAA CAC CCG-31) oligonucleotides, which contain a Kpnl restriction site, are selected by PCR to generate a porA promoter, and Embed in the correct direction (transcription -74· This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ----.----------------^- -------- (Please read the note on the back and fill out this page) Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1297731 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (72 Recombinant pure line according to EcoRI to Xbal direction, and used to transform N. meningitidis B group lacking capsular polysaccharide (cps-) and one of the main outer membrane proteins _PorA (pr〇A-) . A recombinant strain of Neisseria meningitidis produced by double traversal action was selected on g C medium containing 5 μg/ml chloramphenicol (PCR screening using oligonucleotide nucleoside Cam-5 (5,-GTA CTG CGA TGA) GTG GCA GG-3') and proD15-52) were analyzed for D15/Omp85 performance. As shown in Fig. 10, there was a significant increase in the production of D15/Omp85 in the total protein extract of the Nm strain due to the promoter substitution 'compared with the parental strain (cps-). This result was also observed when analyzing large blisters made of the same strain (see Figure 17). These results can be attributed to the replacement of the endogenous D15 promoter with a strong PorA promoter. Furthermore, it was surprisingly found that when the PorA promoter was introduced about 4 bp upstream of the initiation codon, its performance was about 5 〇 more than that when the promoter was introduced upstream by about 1 bp. In general, these experiments support a promoter replacement strategy that is feasible and can positively regulate the synthesis of intact outer membrane proteins in the outer membrane large blisters. Certain geographically isolated human populations (such as Cuba) are infected with a limited number of N. meningitidis isolates that are mostly of one or less outer membrane protein serotypes. Since PorA is the major outer membrane protein antigen that confers protective and strain-specific bacterial antibodies, it is possible to provide a vaccine protection by using a limited amount of PorA serotype in this population. Furthermore, PorA can interact or stabilize with some other outer membrane proteins. In this condition, the presence of PorA in the adventitial sac may be beneficial to enhance the efficacy of the recombinant in improving the large blister. For this reason, it is hoped that the D15/0lnp85 outer membrane protein can be positively regulated at the -75- paper scale for the Chinese National Standard (CNS) A4 specification (210 X 297 mm).

-L (Please read the note on the back and fill out this page) -----r---订--- MW. Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1297731 A7 --- --B7__ V. Description of the invention (73) Lack of a functional cps gene but exhibiting the performance of PorA meningitidis serogroup B strain of PorA. The gene body was extracted from the recombinant B. meningitidis serum B group eps-, p〇rA-, D15/Omp85+ strain using the QIAGEN Genomic Tips 100-G kit. Ten micrograms of this material was linearized, and then transformed into the serum B group cps- of N. meningitidis using a typical transformation strategy. Recombinant Neisseria gonorrhoeae can be obtained on a GC agar plate containing 5 μg/ml chloramphenicol.

The integration by the double traversal effect upstream of the D15 gene was screened by PCR as previously described. Homogeneous recombination can occur anywhere on the chromosome, and a second round of PCR screening is performed to control the integrity of the p〇rA region in the recombinant strain. Based on this, the internal PorA primer PPA1 (5-GCG GCC GTT GCC GAT GTC AGC C_3') and PpA2 (5_GGC ATA GCT GAT GCG Ding 00 human input (: D(0):-3') can be used for the 〇11 screening experiment. The expansion of the 1170乜 fragment confirmed the presence of the PorA gene in the recombinant strain. Recombinant bacteria (corresponding to approximately 5.108 bacteria) can be resuspended in 50 μl of PAGE-SDS buffer solution and frozen ( _2 〇 ° C) / boil (l 〇〇 ° C) three times, and then separated by 12.5% gel PAGE-SDS electrophoresis. The gel was stained with Coomassie Blue R250, or transferred to the nitrocellulose membrane, It is also detected by anti-PorA monoclonal antibody or anti-D15/Omp85 rabbit polyclonal antibody. Analysis of large cell carcinoma of the outer membrane by the same strain can also be performed. Example 1 1 : Hsf protein antigen lacks functional cps Genes but can express positive regulation in the recombinant group of N. meningitidis serogroup B of PorA. As mentioned previously, in some countries, the presence of P〇rA in the outer membrane sac is beneficial and can be strengthened. The recombinant has improved the effectiveness of the vaccine for large blisters. The following national standards apply to the -76-^ paper scale. CNS)A4 specification (210 X 297 mm) I.—-------. —·——^--------- (Please read the notes on the back and fill out this page) A7 1297731 B7 _ V. INSTRUCTION DESCRIPTION (74) In the example, a modified pCMK(+) vector was used to positively regulate the expression of Hsf protein antigen in a strain lacking a functional cps gene but expressing PorA. The original pCMK The (+) vector contains a chimeric PorA/lacO promoter that is regulated in E. coli on the surface laclq but is transcriptionally active in N. meningitidis. In the modified pCMK(+), native The PorA promoter can be used to drive the transcription of the hsf gene. The gene that directs the synthesis of Hsf is amplified by PCR using the HSF 01-NdeI and HSF 02-NheI oligonucleotide primers shown in the table below. Due to the HSF 01-NdeI primer sequence For this reason, the Hsf protein will exhibit two methionine residues at the 5' end. The conditions for PCR amplification are as described by the supplier (HiFi DNA polymerase, Boehringer Mannheim, GmbH). The thermal cycle is as follows : 2 5 times (94 ° C, 1 minute, 48 ° C, 1 minute, 72 ° C, 3 minutes) and 1 time (72 ° C, 10 minutes, 4 ° C until recovery). The equivalent expander was re-selected to the equivalent position of the PCKK (+) delivery vector. In this recombinant plastid, named pCMK(+)-Hsf, we used recombinant PCR The strategy was to delete the lacO present in the chimeric P〇rA/lacO promoter (see Figure 12). The pCMK(+)-Hsf plastid is used as a template to expand two different DNA fragments by PCR: - and the segment contains the PorA 5f recombinant region, the Kangmycin resistance gene and the PorA promoter. The oligonucleotide primers used, RP1 (SacII) and RP2, are shown in the table below. The sequence upstream of the RP1 primer and the lac operator is homogenous. Fragment 2 contains the S-D sequence from the PorA gene, and the hsf gene and PorA 3' have a recombination region. The oligonucleotide nucleoside primers used are RP3 and RP4 (Apal) shown in the table below. The RP3 primer is homologous to the downstream sequence of the lac operator. The 3' end of fragment 1 and the 5' end of fragment 2 have 48 base overlaps. 500-77 for each PCR - This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) ----_------------r------ ------ # (Please read the notes on the back and fill out this page.) Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1297731 A7 B7 V. Invention Description (75) Nanograms (1 and 2) for The final PCR reaction using primers RP1 and RP4 was used. The resulting expanders were subcultured into PSL 1180 vectors that have been restricted by SacII and Apal. The modified plasmid pCMK(+)-Hsf was purified in a large-scale manner using the QIAGEN maxiprep kit, and 2 μg of this material was transformed into the N. meningitidis serum B group strain lacking the functional cps gene (example) The strain described in 1). In order to maintain the performance of PorA, a combination of PCR and Western blotting screening steps was selected to select the integration effect generated by a single traverse. The pure line of Kangmycin resistance positive by PorA-specific PCR and Western blot test was selected and stored in glycerol stock solution at -70 °C for further study. The bacteria (corresponding to about 5.1 〇8 bacteria) were resuspended in 50 μl of PAGE-SDS buffer solution, frozen (-20 ° C) / boiled (l 〇〇 ° C) three times, and then electrophoresed by PAGE-SDS at 12.5%. Separated on the gel. The expression of Hsf was examined in whole cell bacterial lysate (WCBL) derived from NmB [Cps-, PorA+] or NmB [Cps·, PorA+, Hsf+]. Coomassie blue staining measured a significant increase in Hsf performance (in terms of endogenous Hsf levels) (see Figure 13). This result confirmed that the modified pCMK(+)-Hsf vector was functional and successfully used for the forward regulation of the expression of the outer membrane protein without eliminating the production of the major PorA outer membrane protein antigen. Oligonucleotides used in this study----^------------r---book---------I MW (please read the note on the back first) Matters fill out this page) Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperatives Printed Oligonucleotide Sequences HsfOl-Nde 5f-GGA ATT CCA TAT GAT GAA CAA AAT ATACCGC-31 Ndel Selection Site Hsf02-Nhe 5-GTA GCT AGC TAG CTT ACC ACT GAT AAC CGA C-3' Nhel colonization position-78- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) A7 1297731 __B7 V. Description of invention (76) GFP- mut-Asn 5f-AAC TGC AGA ATT AAT ATG AAA GGA GAA GAA CTT TTC-3* Asnl colonization position compatible with Ndel GFP-Spe 5-GAC ATA CTA GTT TAT TTG TAG AGC Spel colonization position TCATCCATG-3, Compatible with Nbel RP1 (SacII) 5,-TCC CCG CGG GCC GTC TGA ATA CAT CCC GTC-31 SacII Selection Site RP2 5-CTA ATG GGC TTC CTT TTG TAA ATT TGA GGG CAA ACA CCC GAT ACG TCT TCA-3 'RP3 5,-AGA CGT ATC GGG TGT TTG CCC TCA AAT TTA CAA AAG GAA GCC CAT ATG-3f RP4 (Apal) 5,-GGG TAT TCC GGG CCC TTC AGA CGG CGC AGC AGG-3, Apal Colonization Position Example 1 2 : Performance of fluorescent protein in a recombinant B. strain of N. meningitidis serotype B that lacks a functional CPS gene but can express PorA. In the following example, a cytoplasmic heterogeneous protein was tested in N. meningitidis using the pCMK vector. Performance. The green fluorescent protein was expanded from the pKen_Gfpmut2 plastid, and the primers were GFP-Asn-mut2 and GFP-Spe (see Table 1 1). Asnl can generate a mismatch with Ndel, and Spel can generate Nhel-compatible mismatches. The conditions for PCR amplification are as described by the manufacturer (HiFi DNA polymerase, Boehringer Mannheim, GmbH). The thermal cycle is as follows: 25 times (94 ° C, 1 minute, 48 ° C, 1 minute, 72. (:, 3 minutes) and 1 time (72 ° C, 10 minutes, 4 ° C until recovery). The expander was re-colonized in the pCMK(+) delivery vector which had been hydrolyzed with Ndel and Nhel restriction enzymes. In this recombinant plastid, it was named pCMK(+)-GFP, and we deleted -79- this paper size is applicable to China. Standard (CNS) A4 specification (210 X 297 mm) ----^------------Book--------- (Please read the notes on the back and fill in On this page) Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed 1293731 A7 B7 _ V. Invention Description (77) (Please read the note on the back and then fill out this page) to remove the lacO present in the chimeric PorA/lacO promoter. Using recombinant PCR strategy, PCR using pCMK(+)-GFR plastids as a template expands two different DNA fragments: - Fragment 1 contains PorA 5' recombinant region, a taxomycin resistance gene and PorA Promoters. The oligonucleotide primers used were: RP1 (SacII) and RP2 (see Table 11). The RP1 primer is homologous to the sequence upstream of the lac operator. - Fragment 2 contains the PorA SD sequence. The gfp gene and PorA 3' have a recombination region. The oligonucleotides used, RP3 and RP4 (Apal), are shown in the table of Example 1. The RP3 primer is homologous to the sequence downstream of the lac operator. 'The end and the 5' end of fragment 2 have 48 base overlap. Each PCR is used for the final PCR reaction at 500 ng (1 and 2), and primers RP1 and RP4 are used. This PCR fragment takes 20 μg for Transgenic serogroup B strain of N. meningitidis lacking functional cps gene. Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed the linear DNA transformation is not as efficient as cyclic plastid DNA, but all the recombination The sub-period can be double-crossed (confirmed by a combination of PCR and Western blotting steps). The pure line resistant to oxytocin is given at -70 ° C in glycerol stock and can be used in further studies. The bacteria (corresponding to approximately 5.108 bacteria) were resuspended in 50 μl of PAGE-SDS buffer solution, frozen (-20 ° C) / boiled (100 ° C) three times, and electrophoresed on a 12.5% gel by PAGE-SDS. Separation. GFP performance in NmB [Cps_, PorA+] or NmB [Cps-, PorA-, GFP+] Examination in whole-cell bacterial lysate (WCBL). Coomassie staining can detect the presence of GFP in the recipient of meningitis Neisser-80 - This paper scale applies to the Chinese National Standard (CNS) A4 specification ( 210 X 297 mm) 1297731 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed A7 B7___ V. Invention Description (78) Cocci (see Figure 14) Ointment Example 1 3 ··Replacement with promoter, positive regulation of meningitis The purpose of the NspA gene assay of the Neisseria serogroup is to replace the endogenous promoter region of the NspA gene with a strong PorA promoter to positively regulate the production of NspA antigen. For this purpose, promoters were used to construct plastids using the E. coli selection method. A DNA region upstream of the NspA-encoding gene (924 bp) can be found in (SEQ ID NO: 7) a private Incyte PathoSeq library containing the untreated DNA sequence of the N. meningitidis strain ATCC 13090. For the NspA gene initiation codon (ATG), a DNA fragment (675 bp) covering nucleotide-115 to -790 was amplified by PCR using the oligonucleotide PNS1, (5,-CCG CGA ATT CGA CGA AGC) CGC CCT CGA C-3,) and PNS2 (5,-CGT CTA GAC GTA GCG GTA TCC GGC TGC_3'), which contain EcoRI and Xbal restriction positions (underlined), respectively. The PCR fragment was subjected to EcoRI and Xbal restriction hydrolysis and then embedded in pUC18. This plastid undergoes a circular PCR mutation (Jones &amp; Winistofer (1992), Biotechniques 12: 528-534) to embed the meningococcal absorption sequence required for transformation, and a suitable restriction position to enable selection of CmR/PorA Son. Perform circular PCR using BAD01-2 (5f-GGC GCC CGG GCT CGA GCT TAT CGA TGG AAA ACG CAG C-3,) and BAD02-2 (5f-GGC GCC CGG GCT CGA GTT CAG ACG GCG CGC TTA TAT AGT GGA TTA AC-3') oligonucleotide containing a suitable restriction position (Xmal and Xhol) for the absorption sequence and underlined. The resulting PCR fragment was gel purified and hydrolyzed with Xhol. The CmR/PorA promoter is from the previously described pUC D15/Omp85 -81 - This paper size applies to the Chinese National Standard (CNS) A4 specification (210 297 297 mm) ----^-------- ----r---订--------- (Please read the phonetic on the back? Please fill out this page again) A7 1297731 ____B7___ V. Invention description (79) Enlargement in the plastid, then introduce BAD 15-2 (5'-GGC GCC CGG GCT GGA GTC TAG ACA TCG GGC AAA CAC CCG-3,) and BAD 03-2 (5,-GGC GCC CGG GCT CGA GCA CTA GTA TTA CCC TGT TAT CCC-3' An oligonucleotide in which the appropriate restriction positions (Xmal, Xbal, Spel and Xhol) are underlined. The resulting PCR fragment was hydrolyzed and embedded in a circular PCR plastid that had been bound with comparable enzymes. One hundred micrograms of recombinant plastids were linearized and used to transform into a N. meningitidis serotype B strain lacking capsular polysaccharide (cps-) and a major outer membrane protein-PorA (porA-). Selected by double traversal on GC agar containing 5 μg/ml chloramphenicol [PCR screening using oligonucleotides BAD 25 (5f_GAG CGA AGC CGT CGA ACG C-3,) and BAD08 (5,-CTT AAG CGT CGG) ACA TTTCC-3')] The recombinant Neisseria meningitidis strain was generated and analyzed for NspA expression. Recombinant bacteria (corresponding to approximately 5.108 bacteria) were resuspended in 50 μl of PAGE-SDS buffer solution, frozen (-20 ° C) / boiled (100 ° C) three times, and electrophoresed on a 12.5% gel by PAGE-SDS. Separation. The gel was stained with Coomassie Brilliant Blue R250 or transferred to a nitrocellulose membrane and probed with anti-porA monoclonal antibody or anti-NspA monoclonal antibody (Fig. 17). As for 〇mp85, there are striking indications that the promoter is inserted about 400 bp upstream of the NSpA kinetic codon, and its performance is stronger than that placed upstream of about 1 bp. The same recombinant pUC plastid can be used to positively regulate the expression of NspA in the strain B strain of N. meningitidis serum that still lacks the functional cps gene but still exhibits PorA. Wide mover replacement positive regulation of N. meningitidis serum Β type nldA (omplA) a factor -82- This paper scale applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) ----- ---------------Book --------- (Please read the notes on the back and fill out this page) Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperative, Ministry of Printing and Economy Intellectual Property Bureau Staff Consumer Cooperative Printed 1297731 A7 ___Β7 ___ V. INSTRUCTIONS (80) The purpose of the experiment was to replace the endogenous promoter region of the pldA (omplA) gene with a strong porA promoter to positively regulate PldA (OmplAl). ) The production of antigens. For this purpose, a promoter replacement medium was constructed by the E· coli coding method. A DNA region (373 bp) upstream from the pdlA coding sequence can be found in (8 ugly (^10&gt;10:18) private 111〇)^?&amp;1:11〇864 database, Neisseria meningitidis ATCC 13090. This DNA contains a sequence that directs the synthesis of the putative rpsT gene. The stop codon position of rpsT is 169pb upstream of pldA ATG. To avoid the possible disruption of this important gene, we decided to embed the CmR/PorA promoter upstream of the ATG of pldA. For this purpose, the 992 bp DNA fragment corresponding to the rpsT gene, the 169 bp integration original sequence and the first nucleotide of the 499 pldA gene are amplified by PCR from the DNA of the B group of N. meningitidis serum. The absorption sequence (underlined) oligonucleotides PLA1 Amo5 (5f-GCC GTC TGA ATT TAA AAT TGC GCG TTT ACA G-3f) and PLA1 Amo3 (5f-GTA GTC TAG ATT CAG ACG GCG CAA TTT GGT TTC CGC AC- 3,). PLA1 Amo3 also contains an Xbal limit position. This PCR fragment was cleared in the High Pure kit (Roche, Mannheim, Germany) and directly colonized into the pGemT vector (Promega, USA). This plastid accepts a circular PCR mutation (Jones &amp; Winistofer (1992)) to embed a suitable restriction site to select the CmR/PorA promoter. Cyclic PCR shows CIRCl-Bgl (5,-CCT AGA TCT CTC CGC CCC CCA TTG TCG-3,) and CIRC1-XH-RBS/2 (5f-CCG CTC GAG TAC AAA AGG AAG CCG ATA TGA ATA TAC GGA ATA TGC G-3,) or CIRC2-XHO/2 (5,-CCG CTC GAG ATG AAT ATA CGG AAT-3') oligo-nucleic acid, which contains underlined -83- This paper scale applies to Chinese national standards (CNS )A4 size (210 X 297 mm) -------------I -----Γ---订--------- (Please read the back of the note first) Matters fill out this page) 1297731 A7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing _____ B7_____ V. Invention description (81) Suitable restricted position (Bglll and Xhol). The CmR/PorA promoter was expanded from the previously described pUCD15/Omp85 plastid using the primer BAD20 (5'-TCC CCC GGG AGA TCT CAC TAG TAT TAC CCT GTT ATC C-3f) &amp; CM-PORA-3 ( 5丨-CCG CTC GAG ATA AAA ACC TAA AAA CAT CGG GC-3) contains suitable restricted positions (Bglll and Xhol) underlined. This PCR fragment was cloned into the circular PCR plastids obtained with the primers CIRCl-Bgl and CIRC1-XH-RBS/2. This plastid can be used to transform the N. meningitidis serum B group [cps·] and [cps_ porA_] strains. In the upstream region of pldA, the porA promoter can be directly inserted upstream of the pldA ATG via the integration of a double traverse effect. The other sputum was replaced by a promoter, and the genomic DNA of the recombinant B. meningitidis serotype B [cps-, porA-, D15/Omp85+] was expanded. This 匣 contains the cmR gene, 卩 (^ into the promoter and 400 6? is equivalent to 〇15/〇11^85 of 5, contiguous sequence. This sequence has been shown to be positive in the regulation of D15/Omp85 in Neisseria. Efficient and can be used to test the positive regulation of other Neisserial antigen expression. The primers for expansion are B AD 20 and CM-PORA-D15/3 (5丨_CGG CTC GAG TGT CAG TTC CTT GTG GTG C_3,), which contains the Xhol restriction position (underlined). This PCR fragment was cloned into the circular PCR plastid obtained with the primers CIRCl-Bgl and CIRC2-XHO/2. This plastid can be used to transform meningitis. Neisserial serogroup B [cps-] and [cps-, porA_] strains. The integration effect of the double cross in the upstream region of pldA can direct the insertion of the porA promoter by 400 bp upstream of pldA ATG. 1 5 : Replace the positive regulation of Neisseria meningitidis serum b group tbpA gene with promoter (please read the back of the note and fill out this page) Install II aamm —1 set ---------AW -84 - ^The paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1297731 Ministry of Economic Affairs Intellectual Property Office employees The fee cooperative printed A7 __Β7__ V. Inventive Note (82) The purpose of the experiment was to replace the endogenous promoter region of the tbpA gene with a strong porA promoter to positively regulate the production of TbpA antigen. For this purpose, use E. coli The selection method is used to construct a promoter to replace the plastid. A DNA region (713 bp) upstream of the tbpA coding sequence can be found in the private Incyte PathoSeq database of N. meningitidis ATCC 13090. This DNA contains a sequence that directs the synthesis of the TbpB antigen. The gene is organized in the operon. The tbpB gene can be deleted and replaced by the CmR/porA promoter. For this purpose, it is equivalent to the tbpB gene 509 bP 5' contiguous region, 2139 bp tbpB coding sequence, 87 bp its integrin sequence and 483 The 3218 bp DNA fragment of the first nucleotide of the tbpA coding sequence was amplified by PCR from the genomic DNA of the N group of N. meningitidis serum, using the oligonucleotide BAD16 (5^GGC CTA GCT AGG CGT CTG AAG) CGA TTA GAG TTT CAA AAT TTA TTC-3?) and BAD17 (5,-GGC CAA GCT TCA GAC GGC GTT CGA CCG AGT TTG AGC CTT TGC_3') contain absorption sequence and Nhel and Hindlll restricted position ). This PCR fragment was cleaned in a High Pure kit (Boerhinger Mannheim, Germany) and directly colonized into the pGemT vector (Promega, USA). This plastid accepts a circular PCR mutation (Jones &amp; Winistofer (1992)) with (1) insertion into a suitable restriction site for selection into the CmR/PorA promoter, and (ii) detection of a 209 bp tbpB 5» contiguous sequence And tpbB coding sequence. Circular PCR utilizes BAD 18 (5'-TCC CCC GGG AAG ATC TGG ACG AAA AAT CTC AAG AAA CCG-3,) and BAD 19 (5f-GGA AGA TCT CCG CTC GAG CAA ATT TAC AAA AGG AAG CCG ATA TGC AAC AGC AAC ATT TGT TCC G-3') Oligonucleotide, containing suitable restriction positions Xmal, Bglll and -85- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) ----^ ------------r ----Book--------- (Please read the notes on the back and fill out this page) _ 1297731 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed A7 B7 _;_ V. Description of invention (83)

Xhol (underlined). The CmR/PorA promoter was expanded from the previously described pUC D15/Omp85 plastid, using the primers BAD21 (5'_GGA AGA TCT CCG CTC GAG ACA TCG GGC AAA CAC CCG-3f) and BAD20 (5,-TCC CCC GGG AGA) TCT CAC TAG TAT TAC CCT GTT ATC CC-3') contains suitable restriction positions Xmal, Spel, Bglll and Xhol (underlined). This PCR fragment was cloned into a circular PCR plastid: this plastid was used to transform N. meningitidis serogroup B [cps-] and [cps-porA-] strains. In the upstream region of tbpA, the integration of the upstream porA promoter of tbpA ATG can be commanded by the integration effect caused by the double traversal action. Example 1 6: The purpose of replacing the positively regulated N. meningitidis serum B group pil〇 gene by a promoter is to replace the endogenous promoter region of the pilQ gene with a strong porA promoter to positively regulate the pilQ antigen. Production system. For this purpose, promoters were used to construct plastids using the E. coli selection method. The DNA region (772 bp) upstream of the pilQ coding gene can be found in (SEQ ID NO: 1.2) the private Incyte PathoSeq database of N. meningitidis ATCC 13090. This DNA contains sequences that direct the synthesis of the PilP antigen. The pilQ gene is a part of the operon. We don't want to interfere with it. Ciliate is an essential element of bacteria. The CmR/porA promoter was introduced upstream of the pilQ gene by the same strategy as the forward regulation of circulating pldA gene expression. For this purpose, the 3' part of the pilP coding sequence, the 18 bp 866 bp DNA fragment with the integration of the original sequence and the first nuclear acid of the 392 pilQ gene, was amplified by PCR from the DNA of the B group of the Neisserial serogroup B gene. , which utilizes PQ-rec5-Nhe (5'-CTA GCT AGC GCC GTC TGA ACG ACG CGA AGC CAA AGC-3,) -86- No paper scale applies Chinese National Standard (CNS) A4 specification (210 x 297 mm)

_---;---^----^--------^--------- (Please read the notes on the back and fill out this page) ^I Ministry of Economics Property Bureau Staff Consumer Cooperatives Printed 1229731 A7 _____B7___ V. Inventions (84) and PQ-rec3-Hin (GCC AAG CTT TTC AGA CGG CAC GGT ATC GTC CGA TTC G-3') Oligonucleotide, containing absorption sequences and Nhel and Hindlll limit positions (underlined). This PCR fragment was directly colonized into the pGemT vector (Promega, USA). This plastid accepts a circular PCR mutation (Jones & Winistofer (1992)) to be inserted into a suitable restriction site and is allowed to be cloned into the CmR/PorA promoter library. Circular PCR utilizes CIRCl-PQ-Bgl (5'-GGA AGA TCT AAT GGA GTA ATC CTC TTC TTA-3,) and CIRC1-PQ-XH0 (5,-CCG CTC GAG TAC AAA AGG AAG CCG ATA TGA TTA CCA AAC TGA CAA AAA TC-3,) or CIRC2-PQ-X (5f-CCG CTC GAG ATG AAT ACC AAA CTG ACA AAA ATC-3') oligonucleotides with suitable restriction positions Bglll and Xhol (underlined) . The Cmr/PorA promoter was expanded from pUC D15/Omp85, using primers BAD20 (5'-TC CCC GGG AGA TCT CAC TAG TAT TAC CCT GTT ATC CC-3) and CM-PORA-3 (5f-CCG CTC) GAG ATA AAA ACC TAA AAA CAT CGG GCA AAC ACC C-3') contains suitable restricted positions Bglll and Xhol (underlined). This PCR fragment was cloned into the circular PCR plastid obtained with the primers CIRCl-PQ-Bgl and CIRC1-PQ-XHO. This plastid can be used to transform into N. meningitidis serum B group [cps-] and [cps-, porA-] strains. In the upstream region of pilQ, the porA promoter can be directly inserted upstream of the pilQ ATG via the integration of the double cross. The other is to expand the genomic DNA of D15/Omp85 from the recombinant B. meningitidis serum B group [cps-, porA-, D15/Omp85+] and replace it with a promoter. This 匣 contains cmR gene, porA promoter and -87- This paper scale applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm) ----^------------r ---Book---------- (Please read the phonetic on the back? Please fill out this page again) 1297731 A7 ___ B7 _ V. Invention description (85 ) (Please read the notes on the back and fill in the form This page) 400 bp is equivalent to the D15/Omp85 gene 5, contiguous sequence. This sequence has been shown to be effective in positively regulating D15/Omp85 expression in N. meningitidis and can be used to test positive regulation of other Neisserial antigen expression. The primers for the amplification were BAD 20 and CM-PORA-D153 (5, · GGG CTC GAG TGT CAG TTC CTT GTG GTG C-3,), which contained Xbol restriction positions (underlined). This PCR fragment was cloned into the circular PCR plastid obtained with the primers CIRCl-PQ-Bgl and CIRC2-PQ-X. This plasmid can be used to transform N. meningitidis serum B group [cps_] and [cps-, porA-] strains. The double traversal effect in the upstream region of pilQ can be used to instruct the pilQ ATG 400 bp upstream and the porA promoter to be inserted. Example 1 7: Construction of kanR/sacB匣 to introduce “cleansing” into the N. meningitidis chromosome. The purpose of the unlabeled mutation experiment was to construct a diverse DNA containing a selectable marker to screen the meninges positively. Recombination in the chromosome of N. sphaeroides (ie, the kanR gene), and a relative screenable marker to delete the sputum in the recombined chromosome (ie, the sacB gene). This method is introduced in homogenous recombination. The heterogeneous DNA can be removed from the chromosome of Neisseria. The Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative, prints the PIB 279 plastid with BamHI restriction enzymes (Blomfield IC, Vaughn V, Rest RF, Eisenstein BI (1991), Mol Microbiol 5:1447-57) A DNA fragment containing the neoR gene and the sacB gene under the control of its own promoter is obtained. The recipient vector is delivered from plastid pCMK (previously described), and the kanR gene of pCMK is restricted by Nrul and EcoRV Deleted as a function. This plastid is named pCMKs°neoR/sacB匣 embedded in pCMKs, and Bglll-restricted position compatible with BamHI end. -88- This paper scale applies to Chinese National Standard (CNS) A4 Specifications (210 X 297 mm) Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1229731 A7 ___Β7_ V. Description of invention (86) E. coli with plastids cannot grow in medium with 2% sucrose, confirming sacB Promoter functionality. This plastid contains a sequence of recombinants that allows sputum to be inserted into the porA region of the B group of N. meningitidis serum. Recombinant Neisseria can contain 200 μg/ml The GC was obtained on the GC agar plate. Unfortunately, the sacB promoter was not functional in N. meningitidis: no difference in growth was observed on the GC agar plate containing 2% sucrose. Construct a new sputum, Contains the sacB gene and is under the control of the kanR promoter. Circular PCR is performed using the plastid pUC4K (Amersham Pharmacia Biotech, USA) as a template and CIRC-Kan-Nco (5'-CAT GCC ATG GTT AGA AAA) ACT CAT CGA GCA TC-3,) and CIRC-Kan-Xba (5,-CTA CTC TAG ATC AGA ATT GGT TAA TTG GTT G-3') Oligonucleotide with Ncol and Xbal restriction positions (underlined) The generated PCR fragment is gel purified. Ncol is hydrolyzed and re-linked to the sacB gene, which is composed of the pIB279 plastid with SAC/NCO/NEW5 (5,-CAT GCC ATG GCA GGA TGA ACG ATG AAC ATC AAAAAGTTTGCAA-3') oligonucleotide (containing the Ncol restriction site, Bottom underlined) and RBS (bold) and SAC/NCO/NEW3 (5,-GAT CCC ATG GTT ATT TGT TAA CTG TTA ATT GTC-3,) oligo-aluminum acid (containing Ncol-restricted position, underlined). The recombinant Ε· coli pure line can be tested for sensitivity on agar plates containing 2% sucrose. The new kanR/sacB can be subcultured into pCMKs and used to transform N. meningitidis serum B group cps-strain. The sucrose sensitivity obtained can be confirmed in Neisseria. The pCMKs plastid can be used to transform the recombinant kanR/SacB Neisser-89 - This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ---------- ---------Book--------- # (Please read the back note and then fill out this page) 1297731 A7 B7 V. Inventions (87) bacteria, to delete The complete sputum in the porA region of the chromosome. A cleaned recombinant Neisserial bacterium was obtained on a G C agar plate containing 2% sucrose. Example 1 8: Using a small recombinant sequence (43 bp) to perform homologous recombination in N. meningitidis staining The objective of the experiment was to use a small recombinant sequence (43 bp) to drive Neisseria Embed, modify or delete in a chromosome. The achievement of this experiment can greatly facilitate further research, which avoids the step of subculture of the E. coli homologous sequence (a 43 bp recombinant sequence can be easily added to the PCR amplification primer). The kanR gene was amplified by PCR from the pUC4K plastid, using the oligonucleotide-Can-PorA-5 (5f-GCC GTC TGA ACC CGT CAT TCC CGC GCA GGC GGG AAT CCA GTC CGT TCA GTT TCG GGA AAG CCA CGT TGT GTC-3) Region containing 43 pb and NmB porA gene 5 'contiguous sequence homozygous (abbreviated) and absorption sequence (underlined) and Kan-PorA-3 (5,-TTC AGA CGG CGC AGC AGG AAT TTA TCG GAA ATA ACT GAA ACC GAA CAG ACT AGG CTG AGG TCT GCC TCG-3·) contains a region of 43 bp homologous to the 3 f contiguous sequence of the NmB porA gene (in bold) and an absorption sequence (underlined). The resulting 1300 bp DNA fragment was re-sequenced into the pGemT vector (Promega, USA). This plastid can be used to transform the N. meningitidis serum B group cps-strain. Recombinant Neisseria gonorrhoeae can be obtained on a GC disk containing 200 μg/ml of oxytetracycline. The integration generated by the dual traversal action in the porA region can be screened by PCR and PPA1 and PPA2 as previously described. Example 19: Immunization with WT and recombinant N. meningitidis large blister to actively protect mice -90 - This paper scale applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm) (please read the back Note: Please fill out this page again) 1T--------- Ministry of Economic Affairs Intellectual Property Bureau Employees Consumption Cooperatives Ministry of Printing and Economy Ministry Intellectual Property Bureau Employees Consumption Cooperatives Printed 1297731 A7 __________ V. Invention Description (88) At the first, at 4 and 28 days, three micrograms (IP path) were immunized with 5 micrograms of OMVs adsorbed in A1(〇h)3. On day 28 (14 days after the second dose) and 35 days (7 days after the third), challenge (jp) was given on day 33. The challenge dose is 20 X LD50 (~107 CFU/mouse). The mortality rate was followed up to 7 days after the challenge. OMVs were injected as: Group 1: Cps-, PorA+ large water cancer Group 2: Cps-, PorA-large water cancer Group 3: Cps-, PorA-, NspA+ large blisters Group 4·· Cps-, PorA- , Omp85 + Large Water Cancer Group 5: Cps_, PorA-, Hsf + Large Water Cancer Figure 15 illustrates the format of these OMVs by analyzing SDS Page (Coomassi Blue staining). After 24 hours of challenge, there was a 100% mortality rate (8/8) in the negative control group (immunized with ai(〇h)3 alone), while mice immunized with 5 different OMVs were still alive (7- 8/8 mice survived). The disease was also tracked in 7 days, and the mice immunized with the large vesicles overexpressed by NSPA appeared to be less likely to be sick than the other groups. The presence of p〇rA in P〇rA+ large blisters appears to provide expanded protection against homologous strain infection. However, the protective effect induced by P〇rA_ positively regulated large water cancer appears to be at least to some extent attributable to the increased NspA, Omp85 or Hsf. Rabbit Example 20: Detection by whole-cell ELISA* The immunogenicity of D. sylvestris is also the ability to detect antibodies to confirm the antigen present on the surface of MenB cells, using whole-cell ELISA (using paramycin Inactivated cells) to detect the collected -91 paper size standard time national standard (CNS) A4 specification (210 X 297 mm)

Lr---------------r---book--------- (please read the notes on the back and fill out this page) 1297731 A7 ___B7 V. Invention Description (89) Rat serum (see Example 119), titer expressed as midpoint titer. Each type of large blister antibody produced a high whole-cell A b titer, while the negative control group was clearly negative. Large water cancer WCE (mid point 14P2 H44/76) valence 14P3 CPS(-) PorA (+) 23849 65539 CPS(-) PorA (-) 20130 40150 CPS(-) PorA (-) NSPA(+) 8435 23846 CPS (-) PorA (-) OMP85(+) 4747 16116 CPS(-) PorA (-) HSF(+) 6964 22504 (-) 51 82 (Please read the notes on the back and fill out this page) Loading·--- ----Book--------- Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed the specific Ab reaction analysis of the recombinant HSF cells that can be used. The microplate was coated with a full length of HSF molecules at 1 μg/ml. The results shown in Fig. 16 show that when the OMVs expressed in excess of HSF are immunized to mice (using purified recombinant HSF on the plate), there is a good and specific HSF response. Large blisters with excessive HSF can produce good specific antibody levels. -92- This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) [297731 A7 B7 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed 5, Invention Description (9〇) SEQ. ID NO: l Nuclear station acid sequence of pCMK(+) vector

TCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGT

AATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTA

AAAAGGCCGCGTTGCTGGCGTTnTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGG

TGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCT

GCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCnTCTCATAGCrCACGCTGTAGGTATCTCA

GTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCrGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGT

AACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGC

GAGGTATGTAGGCGGTGCTACAGAGTrCrTGAAGTGGTGGCCTAACTACGGCrACACTAGAAGAACAGTATTTGGTATCT

GCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGT

GGTTTTTTTGnTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTG

TGACGGTCAGTGGAACGAAAACrCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTXT

TAAATTAAAAATGAAGmTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGT

GAGGCACCTATCTCAGCGATCTGTCTATTTCGrrCATCCATAGTTGCCTGACrCCCCGTCGTGTAGATAACTACGATACG

GGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAA

ACCAGGCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGG

GAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCrC

GTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAG

CGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTrATGGCAGCACTG

CATAATTCTCTTACrGTCATGCCATCCGTAAGATGCrTTTCTGTGACTGGTGAGTACrCAACCAAGTCATTCTGAGAATA

GTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACrTTAAAAGTGC

TCATCATTGGAAAACGrrCTTCGGGGCGAAAACrCTCAAGGATCTTACCGCrGTTGAGATCCAGTTCGATGTAACCCACT

CGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGC

AAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACrCATACTCTTCCTmTCAATATTATTGAAGCAnTATCAGG

GTTATTGTCrCATGAGCGGATACATATTTGAATGTAnTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGA

AAAGTGCCACCTGACGTCTAAGAAACCATrATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCnTCG

TCTCGCGCGTrTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGG

ATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCA

GAGCAGATTGTACTGAGAGTGCACCATAAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAAT

CAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGCCCGAGATAGGGTTGAGTG

TTGTTCCAGTTTGGAACAAGAGTCCACTATrAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGC

GATGGCCCACrACGTGAACCATCACCCAAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAA

AGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGG

GCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGC

GCGTACTATGGTTGCTTTGACGTATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCAT

TCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTrCGCTATTACGCCAGCTGGCGAAAGGGGG

ATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGC

TTGCCGTCTGAATACATCCCGTGATTCCTCAAAAACAGAAAACCAAAATCAGAAACCTAAAATCCCGTCATTCCCGCGCA

GGCGGGAATCCAGTCCGTTCAGTTTCGGTCATTTCCGATAAATTCCTGCTGCTnTCATTTCTAGATTCCCACTTTCGTG

GGAATGACGGCGGAAGGGTnTGGmTTTCCGATAAATTCTTGAGGCATTGAAATTCTAGATrCCCGCCTGCGCGGGAA

TGACGGCTGTAGATGCCCGATGGTCTTTATAGCGGATTAACAAAAATCAGGACAAGGCGACGAAGCCGCAGACAGTACAG

ATAGTACGGAACCGATTCACTTGGTGCTTCAGCACCTrAGAGAATCGTTCTCnTGAGCTAAGGCGAGGCAACGCCGTAC

TTGTTTrTGTTAATCCACTATAAAGTGCCGCGTGTGTTTTTITATGGCGTnTAAAAAGCCGAGACTGCATCCGGGCAGC

AGCGCATCGGCCCGCACGAGGTCTCTGGAGTCGCGAGCATCAAGGGCGAATTCTGCAGGGGGGGGGGGGAAAGCCACGTT

GTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAA

CAGTAATACAAGGGGTGrrATGAGCCATATTCAACGGGAAACGTCTTGCrCGAGGCCGCGATrAAATTCCAACATGGATG -93 - This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) ---^-----------------^----- --il^ew. (Please read the note on the back of the page and then fill out this page) _ 1297731 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (91)

CTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCC

GATGCGCCAGAGTTGTrrCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTG

GCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTrTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGA

TCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTC

CTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATC

ACGAATGAATAACGGTTTGGTTGATGCGAGTGAnTTGATGACGAGCGTAATGGCTGGCCTGITGAACAAGTCTGGAAAG

AAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTrCTCACTTGATAACCTTATTTrrGAC

GAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAA

CTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTrTTTCAAAAATATGGTAtTGATAATCCTGATATGAATAAAT

TGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGGTTAATTGGTTGTAACACTGGCAGAGCATTACGCTG

ACTTGACGGGACGGCGGCrTTGTTGAATAAATCGAACmTGCTGAGTTGAAGGATCAGATCACGCATCTTCCCGACAAC

GCAGACCGTTCCGTGGCAAAGCAAAAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTC

GCTCACTTTCTGGCTGGATGATGGGGCGArrCAGGCCrGGTATGAGTCAGCAACACCTTCrrCACGAGGCAGACCTCAGC

GCCCCCCCCCCCCTGCAGGAGGTCrGGGCTTGAATTGTGTTGTAGAAACACAACGTTTrrGAAAAAATAAGCTATTGTrT

TATATCAAAATATAATC^TTTTTAAAATAAAGGTTGCGGCATTTATCAGATATTTGTTCTGAAAAATGGTTTTTTGCGGG

GGGGGGGGTATAATTGAAGACGTATCGGGTGTTTGCCCGGAATTGTGAGCGGATAACAATTCGATGTTTTTAGGTTTrTA

TCAAATTrACAAAAGGAAGCCCATATGCATCCTAGGCCTATTAATATTCCGGAGTATACGTAGCCGGCTAACGTTAACAA

CCGGTACCTCTAGAACTATAGCTAGCATGCGCAAATTTAAAGCGCTGATATCGATCGCGCGCAGATCTGArTAAATAGGC

GAAAATACCAGCTACGATCAAATCATCGCCGGCGTTGATTATGATTTTTCCAAACGCACTTCCGCCATCGTGTCTGGCGC

TTGGCrGAAACGCAATACCGGCATCGGCAACTACACTCAAATTAATGCCGCCrCCGTCGGTTTGCGCCACAAATTCTAAA

TATCGGGGCGGTGAAGCGGATAGCTTrGTrrTTGACGGCTTCGCCTrCATTCTrTGATTGCAATCTGACTGCCAATCTGC

TrCAGCCCCAAACAAAAACCCGGATACGGAAGAAAAACGGCAATAAAGACAGCAAATACCGTCTGAAAGATTTTCAGACG

GTAITTCGCArmTGGCTrGGTTrGCACATATAGTGAGACCTTGGCAAAAATAGTCTGTTAACGAAATrTGACGCATAA

AAATGCGCCAAAAAATnTCAATTGCCTAAAACCTrCCTAATATTGAGCAAAAAGTAGGAAAAATCAGAAAAGTTTTGCA

TnTGAAAATGAGATTGAGCATAAAATTrTAGTAACCrATGTTATTGCAAAGGTCTCGAArrGTCATTCCCACGCAGGCG

GGAATCTAGTCTGTTCGGTTTCAGTTArrTCCGATAAATTCCTGCTGCGCCGTCTGAAGAATTCGTAATCATGGTCATAG

CTGTTTCCTGTGTGAAArTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGG

TGCCTAATGAGTGAGCTAACrCACATTAATTGCGTTGCGCTCACrGCCCGCnTCCAGTCGGGAAACCTGTCGTGCCAGC

TGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGC SEQ. ID N0: 2 The nuclear acid sequence of the upstream DNA region of the NspA gene in the serum group A Z2491 of N. meningitidis (997 bp)

GGAACCGAACACGCCGTTCGGTCATACGCCGCCGAAAGGTTTGCCGCAAGACGAAGCCGCCCTCGACATCGAAGACGCGG

TACACGGCGCGCTGGAAAGCGCGGGnrrGTCCACTACGAAACATCGGCnTTGCGAAACCAGCCATGCAGTGCCGCCAC

AATTTGAACTACTGGCAGTTCGGCGATTATrTAGGCATAGGCGCGGGCGCGCACGGCAAAATTTCCTATCCCGACCGCAT

CGAGCGCACCGTCCGCCGCCGCCACCCCAACGACTACCTCGCCTTAATGCAAAACCGACCGAGCGAAGCCGTCGAACGCA

AAACCGTCGCCGCCGAAGAnTGCCGTTCGAATTCATGATGAACGCCCTGCGCCTGACCGACGGCGTACCCAGCGCGATG ttgcaggagcgcacgggcgccccgagtgccaaaatcatggcgcaaatcgaaacggcaaggcaaaaaggcctgctggaaac

CGACCCCGCCGTATTCCGCCCGACCGAAAAAGGACGCTTGTTTTTAAACGATTTGCTGCAGTGTTTTTTATAGTGGATTA

ACAAAAACCAGTACGGCGTTGCCTCGCCTTAQCTCAAAGAGAACGATTCTCTAAGGTGCTGAAGCACCAAGTGAATCGGT

TCCGTACTATCTGTACTGTCTGCGGCTTCGTCGCCTTGTCCTGATTTTrGTTAATCCACTATATAAGCGCAAACAAATCG

GCGGCCGCCCGGGAAAACCCCCCCGAACGCGTCCGGAAAATATGCTTATCGATGGAAAACGCAGCCGCATCCCCCGCCGG

GCGTTTCAGACGGCACAGCCGCCGCCGGAAATGTCCGACGCTTAAGGCACAGACGCACACAAAAAACGGTATGCCTGCAC

CTGCAACAATCCGACAGATACCGCTGTmTTCCAAACCGTrTGCAAGTTTCACCCATCCGCCGCGTGATGCCGCCACCA -94- This paper scale applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) - Bu - I —---- — (Please read the back note and fill out this page) - 1297731 A7 B7 V. Description of invention (92)

CCATTTAAAGGCAACGCGCGGGTTAACGGCTTTGCCG

SEQ ID N0:. 3 Neisseria meningitidis serogroup B ATCC13090 strain, D15 / Omp85 gene upstream of the core Chang acid sequence (1000 bp) DNA region of ACCATTGCCGCCCGCGCCGGCTTCCAAAGCGGGGACAAAATACAATCCGTCAACGGCACACCCGTTGCAGATTGGGGCAG CGCGCAAACCGAAATCGTCCTCAACCTCGAAGCCGGCAAAGTCGCCGTCGGGTTCAGACGGCATCAGGCGCGCAAACCGT CCGCACCATCGATGCCGCAGGCACGCCGGAAGCCGGTAAAATCGCAAAAAACCAAGGCTACATCGGACTGATGCCCTTTA AAATCACAACCGTTGCCGGTGGCGTGGAAAAAGGCAGCCCCGCCGAAAAAGCAGGCCTGAAACCGGGCGACAGGCTGACT GCCGCCGACGGCAAACCCATTACCTCATGGCAAGAATGGGCAAACCTGACCCGCCAAAGCCCCGGCAAAAAAATCACCCT GAACTACGAACGCGCCGGACAAACCCATACCGCCGACATCCGCCCCGATACTGTCGAACAGCCCGACCACACCCTGATCG GGCGCGTCGGCCTCCGTCCGCAGCCGGACAGGGCGTGGGACGCGCAAATCCGCCGCAGCTACCGTCCGTCTGTTATCCGC GCATTCGGCATGGGCTGGGAAAAAACCGTTrCCCACTCGTGGACAACCCrCAAATTnTCGGeAAACrAATCAGCGGCAA CGCCTCCGTCAGCCATATTrCCGGGCCGCTGACCATTGCCGACATTGCCGGACAGTCCGCCGAACTCGGCTTGCAAAGTT ATTTGGAATTTTTGGCAGTGGTCAGCATCAGOCTCGGCGTGCTGAACCTGCTGCCCGTCCCCGTTTTGGACGGCGGCCAC CTCGTGTTTTATACTGCCGAATGGATACGCGGCAAACCTTrGGGCGAACGCGTCCAAAACATCGGT TTGCGCTTCGGGCT TGCCCrCATGATGCTGATGATGGCGGTCGCCTTCTTCAACGACGTTACCCGGCTGCTCGGTTAGATTTTACGnTCGGAA TGCCGTCTGAAACCGCATTCCGCACCACAAGGAACTGACA

SEQ.IDNO: 4 N. meningitidis class -Hsf acid sequence upstream of the gene nuclear Chang (1000 bp) DNA region of ATTCCCGCGCAGGCGGGAATCCAGAAACGCAACGCAACAGGAATTTATCGGAAAAAACAGAAACCTCACCGCCGTCATTC ccgcaaaagcgggaatctagaaacacaacgccTgcaggactttatcagaaaaaacagaaaccccaccgccgtcattcccgc AAAAGCGGGAATCCAGACCCGTCGGCACGGAAACTTACCGGATAAAACAGTTTCCTTAGATTCCACGTCCTAGATTCCCG CTTTCGCGGGAATGACGAGATTTTAGATTATGGGAATTTATCAGGAATGATTGAATCCATAGAAAAACCACAGGAATCTA TCAGAAAAAACAGAAACCCCCACCGCGTCATTCCCGCGCAGGCGGGAATCCAGAAACACAACGCGGCAGGACTTTATCGG AAAAAACCGAAACCCCACCGACCGTCATTCCCGCAAAAGTTGqAATCCAAAAACGCAACGCAACAGGAATTTATCGGAAA AAACAGAAACCCCCACCGCGTCATTCCCGCGCAGGCGGGAATCCAGAAACACAACGCAACAGGAATTTATCGGAAAAAAC AGAAACCCCACCGACCGTCATTCCCGCAAAAGCGGGAATCCAGCAACCGAAAAACCACAGGAATCTATCAGCAAAAACAG AAACCCCCACCGACCGTCATTCCCGCGCAGGCGGGAATCCAGAAACACAACGCGGCAGGACTTTATCGGAAAAAACAGAA ACCCCACCGACCGTCATTCCCGCAAAAGCTGGAATCCAAAAACGCAACGCAACAGGAATTTATCGGAAAAAACAGAAACC CCACCGCCGTCATTCCCGCAAAAGCGGGAATCCAGACCCGTCGGCACGGAAACTTACCGGATAAAACAGTTTCCTTAGAT TCCACGTCCCAG ATTCCCGCCTTCGCGGGAATGACGAGATTTTAAGTTGGGGGAATTTATCAGAAAACCCCCAACCCCCA AAAACCGGGCGGATGCCGCACCATCCGCCCCCAAACCCCGATTTAACCATTCAAACAAACCAAAAGAAAAAACAAA (Please read the phonetic on the back first? Matters fill out this page) -ΦΜ. 订!!0. Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed SE〇. ID NO: 5 Neisseria meningitidis PilQ gene upstream DNA region

Acid sequence (772 bp) GCGATGTCGGGAAGCCTTCTCCCGAATCATTACCCCTTGAGTCGCTGAAAATCGCCCAATCTCCGGAAAACGGCGGCAAT CATGACGGCAAGAGCAGCATCCTGAACCTCAGTGCCATTGCCACCACCTACCAAGCAAAATCCGTAGAAGAGCTTGCCGC AGAAGCGGCACAAAATGCCGAGCAAAAATAACTTACGTTAGGGAAACCATGAAACACTATGCCTTACTCATCAGCTTTCT GGCTCTCTCCGCGTGTTCCCAAGGTTCTGAGGACCTAAACGAATGGATGGCACAAACGCGACGCGAAGCCAAAGCAGAAA TCATACCTTTCCAAGCACCTACCCTGCCGGTTGCGCCGGTATACAGCCCGCCGCAGCTTACAGGGCCGAACGCATTCGAC TTCCGCCGCATGGAAACCGACAAAAAAGGGGAAAATGCCCCCGACACCAAGCGTATTAAAGAAACGCTGGAAAAATTCAG TTTGGAAAATATGCGTTATGTCGGCATTTTGAAGTCTGGACAGAAAGTCTCCGGCTTCATCGAGGCTGAAGGTTATGTCT ACACTGTCGGTGTCGGCAACTATTTGGGACAAAACTACGGTAGAATCGAAAGCATTACCGACGACAGCATCGTCCTGAAC GAGCTGATAGAAGACAGCACGGGCAACTGGGTTTCCCGTAAAGCAGAACTGCTGTTGAATTCTTCCGACAAAAACACCGA ACAAGCGGCAGCACCTGCCGCAGAACAAAATTAAGAAGAGGATTACTCCATT SEQ ID NO: 6 95- Paper This applies China National Standard Scale (CNS) A4 size (210 X 297 mm) 1297731 A7 B7 V. DESCRIPTION OF THE INVENTION (93) Upstream of the Hap gene of Neisseria meningitidis, nuclear station acid in the DNA region Column (1000 bp)

GTGCGGCAAAAAAGAGCAAAAGCCCGCTGTCGATTGCCTGACCGTCCGCGTCCGTAAAATCAGCATAGGTTGCCACGCGC GGCTTGGGCGTTTTCCCACACAAAGCCTCTGCCATCGGCAGCAGGTTTTTCCCCGATATGCGTATCACGCCCACGCCGCC GCGCCCGGGTGCGGTAGCGACTGCCGCAATCGTTGGAACGTTATCCGACATAAAACCCCCGAAAATTCAAAACAGCCGCG ATTATAGCAAATGCCGTCTGAAGTCCGACGGTTTGGCTTTCAGACGGCATAAAACCGCAAAAATGCTTGATAAATCCGTC CGCCTGACCTAATATAACCATATGGAAAAACGAAACACATACGCCTTCCTGCTCGGTATAGGCTCGCTGCTGGGTCTGTT CCATCCCGCAAAAACCGCCATCCGCCCCAATCCCGCCGACGATCTCAAAAACATCGGCGGCGATTTTCAACGCGCCATAG AGAAAGCGCGAAAATGACCGAAAACGCACAGGACAAGGCGCGGCAGGCTGTCGAAACCGTCGTCAAATCCCCGGAGCTTG TCGAGCAAATCCTGTCCGACGAGTACGTGCAAATAATGATAGCCCGGCGTTTCCATTCGGGATCGTTGCCGCCGCCGTCC GACTTGGCGCAATACAACGACATTATCAGCAACGGGGCAGACCGCATTATGGCAATGGCGGAAAAAGAACAAGCCGTCCG GCACGAAACCATACGGCAAGACCAAACCTTCAACAGGCGCGGGCAACTGTACGGCTTCATCAGCGTCATCCTGATACTGC TTTTTGCCGTCTTCCTCGTATGGAGCGGCTACCCCGCAACCGCCGCCTCCCTTGCCGGCGGCACAGTGGTTGCCTTGGCG GGTGCTTTCGTGATTGGAAGAAGCCGAGACCAAGGCAAAAATTAATTGCAAATCCTAGGGCGTGCTTCATATCCGCCCGA ACGCCGAACCGCACATATAGGCACATCC CGCGCGCCGCCGGAAGCGGAAGCCGCGCCCTCCCAAACAAACCCGAATCCCG TCAGATAAGGAAAAATA SEQ. ID NO: 7 Neisseria meningitidis (serogroup B) (ATCC13090), nucleotide sequence of the upstream DNA region of the NspA gene (924 bp)

GGAACCGAACACGCCGTTCGGTCATACGGCGCCGAAAGGTTTGCCGCAAGACGAAGCCGCCCTCGACATCGAAGACGCGG

TACACGGCGCGCTGGAAGGCGCGGGTTTTGTCCACTACGAAACATCGGCTTTTGCGAAACCAGCCATGCAGTGCCGCCAC

AATTTGAACTACTGGCAGTTCGGCGATTATTTAGGCATAGGCGCGGGCGCTCACGGCAAAATTTCCTATCCCGACCGCAT

CGAGCGCACCGTCCGCCGCCGCCACCCCAACGACTACCTCGCCTTAATGCAAAGCCAACCGAGTGAAGCCGTCGAACGCA

AAACCGTTGCCGCCGAAGATTTGCCGTTTGAGTTCATGATGAACGCCCTGCGCCTGACCGACGCGTACCCGCCGCGATGT

TGCAGGAGCGCACGGGCGTACCGAGTGCCAAAATCATGGCGCAAATCGAAACGGCAAGGCAAAAAGGCCTGCTGGAAACC

GACCCCGCCGTATTCCGCCCGACCGAAAAAGGACGCTTGTTTTTAAACGATTTGCTGCAGTGTTTTTTATAGTGGATTAA

CAAAAACCAGTACGGCGTTGCCTCGCCTTAGCTCAAAGAGAACGATTCTCTAAGGTGCTGAAGCACCAAGTGAATCGGTT

CCGTACTATTTGTACTGTCTGCGGCTTCGTCGCCTTGTCCTGATTTTTGTTAATCCACTATATAAGCGCAAACAAATCGG

CGGCCGCCCGGGAAAACCCGCCCCGAACGCGTCCGGAAAATATGCTTATCGATGGAAAACGCAGCCGCATCCCCCGCCGG

GCGTTTCAGACGGCACAGCCGCCGCCGGAAATGTCCGACGCTTAAGGCACAGACGCACACAAAACCGTATGCCTGCACCT

GCAACAATCCGACAGATACCGCTGTTTTTTCCAAACCGTTTGCA SEQ.IDNO:8 Nucleic acid sequence of the upstream DNA region of the FrpB gene of N. meningitidis (serum B group) (1000 bp)

AAGTGGGAATCTAAAAATGAAAAGCAACAGGAATTTATCGGAAATGACCGAAACTGAACGGACTGGATTCCCGCTTTCGC

GGGAATGACGGCGACAGGGTTGCTGTTATAGTGGATGAACAAAAACCAGTACGTCGTTGCCTCGCCTTAGCTCAAAGAGA

ACGATTCTCTAAGGTGCTGAAGCACCAAGTGAATCGGTTCCGTCCTATTTGTACTGTCTGCGGCTTCGTCGCCTTGTCCT

GATTTCTGTTCGTTTTCGGTTATTCCCGATAAATTACCGCCGTTTCTCGTCATTTCTTTAACCCTTCGTCATTCCCGCGC

AGGCGGGAATCTAGTTTTTTTGAGTTCCAGTTGTTTCTGATAAATTCTTGCAGCTTTGAGTTCCtAGATTCCCACTTTCG

TGGGAATGACGGTGGAAAAGTTGCCGTGATTTCGGATAAATTTTCGTAACGCATAATTTCCGTTTTACCCGATAAATGCC

CGCAATCTCAAATCCCGTCATTCCCCAAAAACAAAAAATCAAAAACAGAAATATCGTCATTCCCGCGCAGGCGGGAATCT

AGACCTTAGAACAACAGCAATATTCAAAGATTATCTGAAAGTCCGAGATTCTAGATTCCCACTTTCGTGGGAATGACGAA

TTTTAGGTTTCTGTTTTTGGTTTTCTGTCCTTGCGGGAATGATGAAATTTTAAGTTTTAGGAATTTATCGGAAAAAACAG

AAACCGCTCCGCCGTCATTCCCGCACAGGCTTCGTCATTCCCGCGCAGGCTTCGTCATTCCCGCATTTGTTAATCCACTA

TATTCCCGCCGTTTTTTACATTTCCGACAAAACCTGTCAACAAAAAACAAGACTTCGCAAATAAAAACGATAATCAGCTT

TGCAAAAATCCCCCCCCCCTGTTAATATAAATAAAAATAATTAATTAATTATTTTTCTTATCCTGCCAAATCTTAACGGT

TTGGATTTACTTCCCTTCATACACTCAAGAGGACGATTGA SEQ. ID NO: 9 Nuclear citrate sequence of the upstream DNA region of the FrpA gene of N. meningitidis (serum B group) (1000 bpj

CTATAAAGATGTAAATAAAAATCTCGGTAACGGTAACACTTTGGCTCAGCAAGGCAGCTACACCAAAACAGACGGTACAA

CCGCAAAAATGGGGGATTTACTTTTAGCAGCCGACAATCTGCACAGCCGCTTCACGAACAAAATGCTATCCATTAGCCAT

GTTCGGGAAAACACGATTTCCCCGTTTGTTTTAGGCTGTCTAAACAAATAACCATAAATGTATATCATTATTTAAAATAA

ATAAAAGTATTTAACTATTATTGACGAAATTTTAGAGAAAGAGTAGACTGTCGATTAAATGACAAACAATAGTGAGAAAG

GAAATATTTACTATCCGAGCACAGAGCATATTTTAGGTAGCCTGTAACTGTTCCTGCTGGCGGAAGAGGATGAAGGTGGA

CTTACCCGAGAATAAATGTCCTGTTGTGTGATATGGATGCCATGCCGCGAAGCAATTGATGCAATCACGGCAGTCCTACT

TGAATGAAACCTGTCGTTGCAGAATTTGAAAACGCTATTTTTAAGAAAGGATAAAGGGAGAAAGAATTTTTGGTTTTTAA

GCTGCATGAAACCGTGTTGGAATAAATGCACACCTACGATAATTAATAATTTTCGTTTTTTATTCTACAAGCTATTTATA

TATGATTGCTAAAAGTTTATTTTTTAGATGCCAAAAAATATATTTTATATACTTCATATTGTTTATATGTCTTTATTTGA

ATATATCTTACGATGGGGAAATATTTATATATTTTATAATAAATTTTACTCATTTGCTAATATGTCATGGAATATTACTT

GTATTTTGTAGAATTTTTCCATATGAAAATATTCCATTTACTATTTTTCTGAACTTTATTAGTTTATTTTTAATATTTTT -96- This paper size is applicable to China National Standard (CNS) A4 specification (210 x 297 mm) (please read the note on the back of the page and then fill in this page) ▼Install-----r---book- -------- Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1297731 A7 B7 V. Description of invention (94)

ACCTCTTATATTTACCATAAGAGAGCTAATTGATTCATATTATATTGAGTCGATAATTAATTTATTCTTAATTTTAATTC CTCACGTTATTTTTTTAATTTACTTGAAAGGAAAGCAGAT SEO. ID NO: 10 'Nuclear acid sequence of the upstream DNA region of the FrpC gene of Neisseria meningitidis (serum B group) (1000 bp)

GGAAACAGAGAAAAAAGTTTCTCTTCTATCTTGGATAAATATATTTACCCTCAGTTTAGTTAAGTATTGGAATTTATACC TAAGTAGTAAAAGTTAGTAAATTATTTTTAACTAAAGAGTTAGTATCTACCATAATATATTCTTTAACTAATTTCTAGGC TTGAAATTATGAGACCATATGCTACTACCATTTATCAACTTTTTATTTTGTTTATTGGGAGTGTTTTTACTATGACCTCA TGTGAACCTGTGAATGAAAAGACAGATCAAAAAGCAGTAAGTGCGCAACAGGCTAAAGAACAAACCAGTTTCAACAATCC CGAGCCAATGACAGGATTTGAACATACGGTTACATTTGATTTTCAGGGCACCAAAATGGTTATCCCCTATGGCTATCTTG cacggtatacgcaagAcaatgccacaaaatggctttccgacacgcccgggcaggatgcttactccattaatttgatagag ATTAGCGTCTATTACAAAAAAACCGACCAAGGCTGGGTTCTTGAGCCATACAACCAGCAAAACAAAGCACACTTTATCCA ATTTCTACGCGACGGTTTGGATAGCGTGGACGATATTGTTATCCGAAAAGATGCGTGTAGTTTAAGTACGACTATGGGAG AAAGATTGCTTACTTACGGGGTTAAAAAAATGCCATCTGCCTATCCTGAATACGAGGCTTATGAAGATAAAAGACATATT CCTGAAAATCCATATTTTCATGAATTTTACTATATTAAAAAAGGAGAAAATCCGGCGATTATTACTCATCGGAATAATCG AATAAACCAAACTGAAGAAGATAGTTATAGCACTAGCGTAGGTTCCTGTATTAACGGTTTCACGGTACAGTATTACCCGT 3CAGCAGCTCACACAGCAGGAGTTGGTAGGTTATCACCAACAAGTAGAGCAATTGGTACAGAGTTTT ATAAAAAATAATTTAAAGGATCTTATT

TTATTCGGGAAAAGC GTAAACAATTCAAAT Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing

SEO.ID NO: 11 Neisseria meningitidis (serogroup B), the area upstream of nuclear DNA genes 〇mp85: y: acid sequence (1〇〇〇 bp) ACGTCCGAACCGTGATTCCGCAACGCCGCGCCCAAAACCAAAGCCCAAGCCAAAATGCCGATATAGTTGGCATTGGCAAT CGCGTTAATCGGGTTGGCGACCAGGTTCATCAGCAGCGATTTCAACACTTCCACAATGCCGGAAGGCGGCGCGGCGGACA CATCGCCCGCGCCCGCCAAAACAATGTGCGTCGGGAAAACCATACCGGCGATGACGGCGGTCAGGGCTGCGGAAAACGTA CCAATGAGGTAAAGGATGATAATCGGCCTGATATGCGCCTTGTTGCCTTTTTGGTGCTGCGCGATTGTGGCCGCCACCAA AATAAATACCAAAACCGGCGCGACCGCTTTGAGCGCGCCGACAAACAGGCTGCCGAACAAGCCTGCCGCCAAGCCCAGTT GCGGGGAAACCGAACCGATTACGATGCCCAACGCCAAACCGGCGGCAATCTGCCTGACCAGGCTGACGCGGCCGATCGCA TGAAATAAGGATTTGCCGAACGCCATAATTCTTCCTTATGTTGTGATATGTTAAAAAATGTTGTATTTTAAAAGAAAACT CATTCTCTGTGTTTTTTTTATTTTTCGGCTGTGTTTTAAGGTTGCGTTGATTTGCCCTATGCAGTGCCGGACAGGCTTTG CTTTATCATTCGGCGCAACGGTTTAATTTATTGAACGAAAATAAATTTATTTAATCCTGCCTATTTTCCGGCACTATTCC GAAACGCAGCCTGTTTTCCATATGCGGATTGGAAACAAAATACCTTAAAACAAGCAGATACATTTCCGGCGGGCCGCAAC CTCCGAAATACCGGCGGCAGTATGCCGTCTGAAGTGTCCCGCCCCGTCCGAACAACACAAAAAC . AGCCGTTCGAAACCCT GTCCGAACAGTGTTAGAATCGAAATCTGCCACACCGATGCACGACACCCGTACCATGATGATCAAACCGACCGCCCTGCT CCTGCCGGCTTTATTTTTCTTTCCGCACGCATACGCGCCT SEQ ID NO: 12 'Neisseria meningitidis (serogroup B) (ATCC13090) gene in nuclear PilQ taste acid sequence (772 bp) DNA region upstream of GCGATGTCGGGAAGCCTTCTCCCGAATCATTACCCCTTGAGTCGCTGAAAATCGCCCAATCTCCGGAAAACGGCGGCAAT CATGACGGCAAGAGCAGCATCCTGAACCTCAGTGCCATTGCCACCACCTACCAAGCAAAATCCGTAGAAGAGCTTGCCGC AGAAGCGGCACAAAATGCCGAGCAAAAATAACTTACGTTAGGGAAACCATGAAACACTATGCCTTACTCATCAGCTTTCT GGCTCTCTCCGCGTGTTCCCAAGGTTCTGAGGACCTAAACGAATGGATGGCACAAACGCGACGCGAAGCCAAAGCAGAAA TCATACCTTTCCAAGCACCTACCCTGCCGGTTGCGCCGGTATACAGCCCGCCGCAGCTTACAGGGCCGAACGCATTCGAC TTCCGCCGCATGGAAACCGACAAAAAAGGGGAAAATGCCCCCGACACCAAGCGTATTAAAGAAACGCTGGAAAAATTCAG TTTGGAAAATATGCGTTATGTCGGCATTTTGAAGTCTGGACAGAAAGTCTCCGGCTTCATCGAGGCTGAAGGTTATGTCT ACACTGTCGGTGTCGGCAACTATTTGGGACAAAACTACGGTAGAATCGAAAGCATTACCGACGACAGCATCGTCCTGAAC GAGCTGATAGAAGACAGCACGGGCAACTGGGTTTCCCGTAAAGCAGAACTGCTGTTGAATTCTTCCGACAAAAACACCGA ACAAGCG . GCAGCACCTGCCGCAGAACAAAATTAAGAAGAGGATTACTCCATT SEQ ID NO: 13 Neisseria meningitidis (serogroup B), the nucleic acid sequence Chang (1000 bp) DNA region upstream of the gene-based -HsF TTTGTTTTTTCTTTTGGTTTGTTTGAATGGTTAAATCGGGGTTTGGGGGCGGATGGTGCGGCATCCGCCCGGTTTTTGGG GGTTGGGGGTTTTCTGATAAATTCCCCCAACTTAAAATCTCGTCATTCCCGCGAAGGCGGGAATCTGGGACGTGGAATCT AAGGAAACTGTTTTATCCGGTAAGTTTCCGTGCCGACGGGTCTGGATTCCCGCTTTTGCGGGAATGACGGCGGTGGGGTT TCTGTTTTTTCCGATAAATTCCTGTTGCGTTGCGTTTTTGGATTCCAGCTTTTGCGGGAATGACGGTCGGTGGGGTTTCT GTTTTTTCCGATAAAGTCCTGCCGCGTTGTGTTTCTGGATTCCCGCCTGCGCGGGAATGACGGTCGGTGGGGGTTTCTGT TTTTGCTGATAGATTCCTGTGGTTTTTCGGTTGCTGGATTCCCGCTTTTGCGGGAATGACGGTCGGTGGGGTTTCTGTTT TTTCCGATAAATTCCTQTTGCGTTGTGTTTCTGGATTCCCGCCTGCGCGGGAATGACGCGGTGGGGGTTTCTGTTTTTTC CGATAAATTCCTGTTGCGTTGCGTTTTTGGATTCCAACTTTTGCGGGAATGACGGTCGGTGGGGTTTCGGTTTTTTCCGA TAAAGTCCTGCCGCGTTGTGTTTCTGGATTCCCGCCTGCGCGGGAATGACGCGGTGGGGGTTTCTGTTTTTTCTGATAGA CATTCCCGCGAAAGCGGG 3GTCTGGATTCCCGCTTTTGCGG GAATGACGGCGGTGGGGTTTCTGTTTTTTCTGATAAAGTCCTGCCGCGTTGTGTTTCTAGATT CCCGCTTTTGCGGGAAT GACGGCGGTGAGGTTTCTQTTTTTTCCGATAAATTCCTGT

TTCCTGTGGTTTTTCTATGGATTCftATCATTCCTGATAAATTCCCATAATCTAAAATCTCGTCA AATCTAGGACGTGGAATCTAAGGAAACTGTTTTATCCGGTAAGTTTCCGTGCCGACGGGTCTGG SEQ. ID NO: 14 Nuclear acid sequence of the upstream DNA region of Hap gene in Neisseria meningitidis (serum B group) (1000 bp) -97- Not paper scale applicable Chinese National Standard (CNS) A4 specification (210 X 297 mm) • installed (read the back of the precautions to fill out this page) ϋ l · - Order -------- 1297731 A7 B7 V. invention is described in (95) AATCAGCATAGGTTGCCACGCGCGGCTTGGGCGTTTTCCCACACAAAGCCTCTGCCATCGGCAGCAGGTTTTTCCCCGAT ATGCGTATCACGCCCACGCCGCCGCGCCCGGGTGCGGTAGCGACTGCCGCAATCGTTGGAACGTTATCCGACATAAAACC CCCGAAAATTCAAAACAGCCGCGATTATAGCAAATGCCGTCTGAAGTCCGACGGTTTGGCTTTCAGACGGCATAAAACCG CAAAAATGCTTGATAAATCCGTCCGCCTGACCTAATATAACCATATGGAAAAACGAAACACATACGCCTTCCTGCTCGGT ATAGGCTCGCTGCTGGGTCTGTTCCATCCCGCAAAAACCGCCATCCGCCCCAATCCCGCCGACGATCTCAAAAACATCGG CGGCGATTTTCAACGCGCCATAGAGAAAGCGCGAAAATGACCGAAAACGCACAGGACAAGGCGCGGCAGGCTGTCGAAAC CGTCGTCAAATCCCCGGAGCTTGTCGAGCAAATCCTGTCCGACGAGTACGTGCAAATAATGATAGCCCGG CGTTTCCATT CGGGATCGTTGCCGCCGCCGTCCGACTTGGCGCAATACAACGACATTATCAGCAACGGGGCAGACCGCATTATGGCAATG GCGGAAAAAGAACAAGCCGTCCGGCACGAAACCATACGGCAAGACCAAACCTTCAACAGGCGCGGGCAACTGTACGGCTT CATCAGCGTCATCCTGATACTGCTTTTTGCCGTCTTCCTCGTATGGAGCGGCTACCCCGCAACCGCCGCCTCCCTTGCCG GCGGCACAGTGGTTGCCTTGGCGGGTGCTTTCGTGATTGGAAGAAGCCGAGACCAAGGCAAAAATTAATTGCAAATCCTA GGGCGTGCTTCATATGCGCCCGAACGCCGAACCGCACATATAGGCACATCCCGCGCGCCGCCGGA CTCCCAAACAAACCCGAATCCCGTCAGATAAGGAAAAATA SEO ID NO:. 15 Neisseria meningitidis (serogroup B), upstream of the gene LbpA

3AAGCGGAAGCCGCGCC DNA region of the nucleotide sequence (1000 bp)

GATTTTGGTCATCCCGACAAGCTTCTTGTCGAAGGGCGTGAAATTCCTTTGGTTAGCCAAGAGAAAACCATCAAGCTTGC CGATGGCAGGGAAATGACCGTCCGTGCTTGTTGCGACTTTTTGACCTATGTGAAACTCGGACGGATAAAAACCGAACGCC CGGCAAGTAAACCAAAGG ZCGAAi CCAAAl CCTCGGAAGCI Ministry of Economic Affairs Intellectual Property Office employees consumer cooperatives printed; GCGGAAGATAAAAGGGAGGATGAAGAGAGTGCAGGCGTTGGTAACGTCGAAGAAGGCGAAGGC iAAGAACCCGfl IAAAAATCGCCGACAGAAGAAAGCGGCAGCGGTTCAAACGCCATCCTGCCTG CTCTAAAGGCAGGGACATCGACCTTTTCCTGAAAGGTATCCGCACGGCGGAAGCCGACATTCCAAGAACC GGAAAAGCACACTATACCGGCACTTGGGAAGCGCGTATCGGCAQAGCCATTCAATGGGACAATCAGGCGGATAAAGAAGC GGCAAAAGCAGAATTTACCGTTAATTTCGGCGAGAAATCGATTTCCGGAACGCTGACGGAGAAAAACGGTGTACAACCTG CTTTCTATATTGAAAACGGCAAGATTGAGGGCAACGGTTTCCACGCAACAGCACGCACTCGTGAGAACGGCATCAATCTT TCGGGAAATGGTTCGACCAACCCCAGAACCTTCCAAGCTAGTGATCTTCGTGTAGAAGGAGGATTTTACGGCCCGCAGCG GAGGAATTGGGCGGTATTATTTTCAATAAGGATGGGAAATCTCTTGGTATAACTGAAGGTACTGAAAATAAAGTTGAAGT TGAAGCTGAAGTTGAAGTTGAAGCTGAAACTGGTGTTGTCGAACAGTTAGAACCTGATGAAGTTAAACCCCAATTCGGCG TGGTATTCGGTGCGAAGAAAGATAATAAAGAGGTGGAAAA SEQ.IDNO: 1 6 N. meningitidis (serum group A), the nucleic acid sequence Ru (1〇〇〇 bp) DNA region upstream of the gene LbpB CGGCGTTAGAGTTTAGGGCAGTAAGGGCGCGTCCGCCCTTAGATCTGTAAGTTACGATTCCGTTAAATAACTTTTACTGA CTTTGAGTTTTTTGACCTAAGGGTGAAAGCACCCTTACTGCTTAAAGTCCAACGACAAAAACCAAAAGACAAAAACACTT .TTATTACCCTAAAATCGAACACCCATAAATGACCTTTTTTGTCTTTGGCGAGGCGGCAGTAAGGGCGCGTCCGCCCTTAG ATCTGTAAGTTATGATTCCGTTAAATAGCCTTTACTGACTTTGAGTTTTTTGACCTAAGGGCGGACGCGCCCTTACTGCT TCACCTTCAATGGGCTTTGAATTTTGTTCGCTTTGGCTTGCTTGACCTAAGGGTGAAAGCACCCTTACTGCCGCCTCGCC AAAGACGAAAAGGGTTATTTACGGGGGTTGGATTTTAGGCAGTAAGGGCGCGTCCGCCCTTAGATCTGTAAGTTATGATT CCGTTAAATAGCCTTTACTGACTTTGAGTTTTTTGACCTAAGGGTGAAAGCACCCTTACTGCTTCACCTTCAATGGGCTT TGAATTTTGTTCGCTTTGGCTTGCTTGATCTAAGGGTGAAAGCACCCTTACTGCCGTCTCGCCGAAGACAACGAGGGCTA TTTAdGGCGTTAGAGTTTAGGGCAGTAAGGGCGCGTCCGCCCTTAGATCCAGACAGTCACGCCTTTGAATAGTCCATTTT GCCAAAGAACTCTAAAACGCAGGACCTAAGGGTGAAAGCACCCTTACTGCCTTACATCCAAGCACCCTTACTGCACCACG TCCACGCACCCTTACTGCCCTACGTCCACGCACCCTTACTGCCCTACATCCAAGCACCCTTACTGCCTTACATAGACAT G ACAGACGCCGAGCAGCGGAACAGGACTAAAAACAATTAAGTGATATTTTTGCCCAACTATAATAGACATGTATAATTATA TTACTATTAATAATAATTAGTTTATCCTCCTTTTCATCCC SEQ.IDNO: 17 Neisseria meningitidis (serogroup B) (ATCC13090), the core region of DNA upstream of seeking acid sequence TbpA gene bad lj (73_l bp) ______, TATGAAGTCGAAGTCTGCTGTTCCACCTTCAATTATCTGAATTACGGAATGTTGACGCGC AAAAACAGCAAGTCCGCGATGCAGGCAGGAGAAAGCAGTAGTCAAGCTGATGCTAAAACG GAACAAGTTGGACAAAGTATGTTCCTCCAAGGCGAGCGCACCGATGAAAAAGAGATTCCA AACGACCAAAACGTCGTTTATCGGGGGTCTTGGTACGGGCATATTGCCAACGGCACAAGC TGGAGCGGCAATGCTTCCGATAAAGAGGGCGGCAACAGGGCGGACTTTACTGTGAATTTC GGTACGAAAAAAATTAACGGCACGTTAACCGCTGACAACAGGCAGGCGGCAACCTTTACC ATTGTGGGCGATATTGAGGGCAACGGTTTTTCCGGTACGGCGAAAACTGCTGACTCAGGT TTTGATCTCGATCAAAGCAATAACACCCGCACGCCTAAGGCATATATCACAAACGCCAAG GTGCAGGGCGGTTTTTACGGGCCCAAAGCCGAAGAGTTGGGCGGATGGTTTGCCTATTCG GACGATAAACAAACGAAAAATGCAACAGATGCATCCGGCAATGGAAATTCAGCAAGCAGT GCAACTGTCGTATTCGGTGCGAAACGCCAAAAGCCTGTGCAATAAGCACGGTTGCCGAAC AATCAAGAATAAGGCCTCAGACGGCACCGCTCCTTCCGATACCGTCTGAAAGCGAAGAGT AGGGAAACACT S . EQ ID NO: 18 Neisseria meningitidis (serogroup B) (ATCC13090), the nucleic acid sequence Chang (373 bp) DNA region upstream of the gene OmplA, CGTACCGCATTCCGCACTGCAGTGAAAAAAGTATTGAAAGCAGTCGAAGCAGGCGATAAAGCTGCCGCACAAGCGGTTTA CCAAGAGTCCGTCAAAGTCATCGACCGCATCGCCGACAAGGGCGTGTTCCATAAAAACAAAGCGGCTCGCCACAAAACCC GTTTGTCTCAAAAAGTAAAACCTTGGCTTGATTTTTGCAAAACCTGCAATCCGGTTTTCATCGTCGATTCCGAAAACCCC TGAAGCCCGACGGTTTCGGGGTTTTCTGTATTGCGGGGACAAAATCCCGAAATGGCGGAAAGGGTGCGGTTTTTTATCCG AATCCGCTATAAAATGCCGTCTGAAAACCAATATGCCGACAATGGGGGTGGAG -98 - GATGAAGGCGAAGAAGCC TGAACCCAAAGAAGTTGAAGAAACCGAAGfl ____ I-. ---.----装---- -----订---------^9. (Please read the phonetic 2 on the back and fill in this page) _ This paper size applies China National Standard (CNS) A4 Specification (210 x 297 mm) 1297731 A7 B7 V. Description of Invention (96)

. SEQ ID NO: 19 Neisseria meningitidis (serogroup B), for Nuclear blanket sequence (1000 bp) DNA region upstream of the gene Plal TTTTGGCTTCCAGCGTTTCATTGTTTTCGTACAAGTCGTAAGTCAGCTTCAGATTGTTGG CTTTTTTAAAGTCTTCGACCGTACTCTCATCAACATAGTTCGACCAGTTGTAGATGrrCA GAGTATCGGTGGCAGCGGCTTCGGCATTGGCAGCAGACGCAGCGTCrGCTTGAGGTTGCA CGGCGTnTTTTCGCTGCCGCCGCAGGCTGCCAGAGACAGCGCGGCCAAAACGGCTAATA CGGATTrTTTCATACGGGCAGATTCCTGATGAAAGAGGTTGGAAAAAAAGAAATCCCCGC GCCCCATCGTTACCCCGGCGCAAGGTTTGGGCATTGTAAAGTAAATTTGTGCAAACTCAA rCCTAAAAAArTATCCTGTTTCCAAAA (

AGCGATATTGGACTGAmTC MTATCCTG,

.GGGGAGAAAAACGT

CCGCCCGATTTTGCCGTTTTTTTGCGCTGTCAGGGTGTCCGACGGGCGGATAGAGAGAAA AGGCTTGCATATAATGTAAACCCCCTTTAAAATTGCGCGTTTACAGAAnTATTTTTCTT CCAGGAGATTCCAATATGGCAAACAGCGCACAAGCACGCAAACGTGCCCGCCAGTCCGTC AAACAACGCGCCCACAATGCTAGCCTGCGTACCGCATTCCGCACCGCAGTGAAAAAAGTA rrGAAAGCAGTCGAAGCAGGCGATAAAGCTGCCGCACAAGCGGTTTACCAAGAGTCCGTC AAAGTCATCGACCGCATCGCCGACAAGGGCGTGTTCCACAAAAACAAAGCGGCACGCCAC AAAAGCCGTCTGTCTGCAAAAGTAAAAGCCTTGGCTTGATTTTTGCAAAACCGCCAAGGC GGTTGATACGCGATAAGCGGAAAACCCTGAAGCCCGACGGTTTCGGGGTTrTCTGTATTG CGGGGGCAAAATCCCGAAATGGCGGAAAGGGTGCGATTTTTTATCCGAATCCGCTATAAA ATGCCGTTTGAAAACCAATATGCCGACAATGG

.CCAATATGCC

rGGGGGCGGAG Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing

SEQ ID NO:. 20: Neisseria meningitidis (serogroup B), the nucleic acid sequence Da (1000 bp) a DNA region upstream of the FhaB gene TACGGAAACTGCAAGCGGATCCAGAAGTTACAGCGTGCATTATTCGGTGCCCGTAAAAAAATGGCTGTTTTCTTTTAATC ACAATGGACATCGTTACCACGAAGCAACCGAAGGCTATTCCGTCAATTACGATTACAACGGCAAACAATATCAGAGCAGC CTGGCCGCCGAGCGCATGCTTTGGCGTAACAGACTTCATAAAACTTCAGTCGGAATGAAATTATGGACACGCCAAACCTA TAAATACATCGACGATGCCGAAATCGAAGTGCAACGCCGCCGCTCTGCAGGCTGGGAAGCCGAATTGCGCCACCGTGCTT ACCTCAACCGTTGGCAGCTTGACGGCAAGTTGTCTTACAAACGCGGGACCGGCATGCGCCAAAGTATGCCTGCACCGGAA GAAAACGGCGGCGATATTCTTCCAGGTACATCTCGTATGAAAATCATTACTGCCGGTTTGGACGCAGCCGCCCCATTTAT TTTAGGCAAACAGCAGTTTTTCTACGCAACCGCCATTCAAGCTCAATGGAACAAAACGCCGTTGGTTGCCCAAGATAAAT TGTCAATCGGCAGCCGCTACACCGTTCGCGGATTTGATGGGGAGCAGAGTCTTTTCGGAGAGCGAGGTTTCTACTGGCAG AATACTTTAACTTGGTATTTTCATCCGAACCATCAGTTCTATCTCGGTGCGGACTATGGCCGCGTATTTGGCGAAAGTGC ACAATATGTATCGGGCAAGCAGCTGATGGGTGCAGTGGTCGGCTTCAGAGGAGGGCATAAAGTAGGCGGTATGTTTGCTT ATGATCTGTTTGCCGGCAAGCCGCTTCATAAACCCAAAGGCTTTCAGACGACCAACACCGTTTACGGCTTC AACTTGAAT TACAGTTTCTAACCTCTGAATTTTTTACTGATATTTAGACGGTCTTTCCTTATCCTCAGACCGTCAAACTTTACCTACGT ACTTGGCGCGCAGTACGTTCATCTTCAAAATGGAATAGAC SEQ.IDNO: 21 Neisseria meningitidis (serogroup B), the nucleic acid sequence Chang Lipo02 gene upstream of the DNA region Pi (1000 bp) TTATCTTGGTGCAAAACTTTGTCGGGGTCGGACTGGCTACGGCTTTGGGTTTGGACCCGCTCATCGGTCTGATTACCGGT TCGGTGTCGCTGACGGGCGGACACGGTACGTCAGGTGCGTGGGGACCTAATTTTGAAACGCAATACGGCTTGGTCGGCGC AACCGGTTTGGGTATTGCATCGGCTACTTTCGGGCTGGTGTTCGGCGGCCTGATCGGCGGGCCGGTTGCGCGCCGCCTGA TCAACAAAATGGGCCGCAAACCGGTTGAAAACAAAAAACAGGATCAGGACGACAACGCGGACGACGTGTTCGAGCAGGCA AAACGCACCCGCCTGATTACGGCGGAATCTGCCGTTGAAACGCTTGCCATGTTTGCCGCGTGTTTGGCGTTTGCCGAGAT TATGGACGGCTTCGACAAAGAATATCTGTTCGACCTGCCCAAATTCGTGTGGTGTCTGTTTGGCGGCGTGGTCATCCGCA ACATCCTCACTGCCGCATTCAAGGTCAATATGTTCGACCGCGCCATCGATGTGTTCGGCAATGCTTCGCTTTCGCTTTTC TTGGCAATGGCGTTGCTGAATTTGAAACTGTGGGAGCTGACCGGTTTGGCGGGGCCTGTAACCGTGATTCTTGCCGTACA AACCGTGGTGATGGTTTTGTACGCGACTTTTGTTACCTATGTCTTTATGGGGCGCGACTATGATGCGGCAGTATTGGCTG CCGGCCATTGCGGTTTCGGCTTGG . GTGCAACGCCGACGGCGGTGGCAAATATGCAGTCCGTCACGCATACTTTCGGCGCG TCGCATAAGGCGTTTTTGATTGTGCCTATGGTCGGCGCGTTCTTCGTCGATTTGATTAATGCCGCGATTCTCACCGGTTT TGTGAATTTCTTTAAAGGCTGATTTTCCGCCTTTCCGACAAAGCACCTGCAAGGTTTACCGCCTGCAGGTGCTTTTGCTA TGATAGCCGCTATCGGTCTGCACCGTTTGGAAGGAACATC SEQ ID NO: 22 Neisseria meningitidis (serogroup B), the nucleic acid sequence Chang (1〇〇〇 bp) DNA region upstream of the gene of Tbp2 CCTACTCCACCGATTCCAATATGCTCGGCGCGACCCACGAAGCCAAAGACTTGGAATTTTTGAACTCGGGCATCAAAA, GTCAAACCCATTATGGGCGTTGCCTTTTGGGACGAAAACGTTGAAGTCAGCCCCGAAGAAGTCAGCGTGCGCTTTGAAGA AGGCGTGCCGGTTGCACTGAACGGCAAAGAATACGCCGACCCCGTCGAACTCTTCCTCGAAGCCAACCGCATCGGCGGCC GCCACGGCTTGGGTATGAGCGACCAAATCGAAAACCGCATCATCGAAGCCAAATCGCGCGGCATCTACGAAGCCCCGGGT ATGGCGTTGTTCCACATCGCCTACGAACGCTTGGTGACCGGCATCCACAACGAAGACACCATCGAACAATACCGCATCAA CGGCCTGCGCCTCGGCCGTTTGCTCTACCAAGGCCGCTGGTTCGACAGCCAAGCCTTGATGTTGCGCGAAACCGCCCAAC GCTGGGTCGCCAAAGCCGTTACCGGCGAAGTTACCCTCGAACTGCGGCGCGGCAACGACTACTCGATTCTGAACACCGAA TCGCCCAACCTGACCTACCAACCCGAACGCCTGAGTATGGAAAAAGTCGAAGG TGCGGCGTTTACCCCGCTCGACCGCAT CGGACAGCTCACGATGCGCAACCTCGACATCACCGACACCCGCGCCAAACTGGGCATCTACTCGCAAAGCGGTTTGCTGT CGCTGGGCGAAGGCTCGGTATTACCGCAGTTGGGCAATAAGAAATAAGGTTTGCTGTTTTGCATCATTAGCAACTTAAGG GGTCGTCTGAAAAGATGATCCCTTATGTTAAAAGGAATCCTATGAAAGAATACAAAGTCGTCATTTATCAGGAAAGCCAG TTGTCCAGCCTGTTTTTCGGCGCGGCAAAGGTCAACCCCGTCAATTTCAGCGCGTTCCTCAACAAACAAACCCCCCGAAG

TC (Please read the note on the back and fill out this page) -99 This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1297731 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative prints V. Description of invention (97)

GCTGGCGGGTCGAGACCTTTGCAATAACATAGGTTACTAA SEQ. ID NO: 23 The nucleotide sequence of the DNA region upstream of the P〇rA gene in Neisseria meningitidis (serum B group) (1〇〇〇 bp)

GAATGACAATTCATAAGTTTCCCGAAATTCCAACATAACCGAAACCTGACAATAACCGTAGCAACTGAACCGTCATTCCC

GCAAAAGCGGGAATCCAGTCCGTTCAGTTTCGGTCATTTCCGATAAATGCCTGTTGCTTTTCATTTCTAGATTCCCACTT

TCGTGGGAATGACGGCGGAAGGGTTTTGGTTTTTTCCGATAAATTCTTGAGGCATTGAAATTCCAAATTCCCGCCTGCGC

GGGAATGACGGCTGCAGATGCCCGACGGTCTTTATAGTGGATTAACAAAAATCAGGACAAGGCGACGAGCTGCAGACAGT

ACAGATAGTACGGAACCGATTCACTTAGTGCTTCAGTATCTTAGAGAATCGTTCTCTTTGAGCTAAGGCGAGGCAACGTC

GTACTGGTTTTTGTTCATCCACTATATATGACACGGAAAACGCCGCCGTCCAAACCATGCCGTCTGAAGAAAACTACACA

GATACCGCCGCTTATATTACAATCGCCGCCCCGTGGTTCGAAAACCTCCCACACTAAAAAAGTAAGGAAACCCTATGTCC

CGCAACAACGAAGAGCTGCAAGGTATCTCGCTTTTGGGTAATCAAAAAACCCAATATCCGGCCGAATACGCGCCCGAAAT

TTTGGAAGCGTTCGACAACAAACATCCCGACAACGACTATTTCGTCAAATTCGTCTGCCCAGAGTTCACCAGCCTCTGCC

CCATGACCGGGCAGCCCGACTTCGCCACCATCGTCATCCGCTACATTCCGCACATCAAAATGGTGGAAAGCAAATCCCTG

AAACTCTACCTCTTCAGCTTCCGCAACCACGGCGATTTTCATGAAGACTGCGTCAACATCATCATGAAAGACCTCATTGC

CCTGATGGATCCGAAATACATCGAAGTATTCGGCGAGTTCACACCGCGCGGCGGCATCGCCATTCATCCTTTCGCCAATT

ACGGCAAAGCAGGCACCGAGTTTGAAGCATTGGCGCGTAA SEQ. ID NO: 24. Neisseria meningitidis (serogroup B) PorA promoter region gatatcgaggtctgcgcttgaattgtgttgtagaaacacaacgtttttgaaaaaataagctattgttttatatcaaaata

TAATCATTTTTAAAATAAAGGTTGCGGCATTTATCAGATATTTGTTCTGAAAAATGGTTTTTTGCGGGGGGGGGGGTATA

. ATTGAAGACGTATCGGGTGTTTGCCCGATGTTTTTAGGTTTTTATCAAATTTACAAAAGGAAGCCCAT SEQ ID NO: 25 Neisseria meningitidis (serum group A), the nucleotide sequence (1000 bp) DNA region upstream of the gene P〇rB gttttctgtttttgagggaatgacgggatgtaggttcgtaagaatgacgggatataggtttccgtgcggatggattcgtc attcccgcgcaggcgggaatctagaacgtggaatctaagaaaccgttttatccgataagtttccgtgcggacaagtttgg attcccgcctgcgcgggaatgacgggattttaggtttctaattttggttttctgtttttgagggaatgacgggatgtagg ttcgtaggaatgacgggatataggtttccgtgcggatggattcgtcattcccgcgcaggcgggaatctagaccttagaac aacagcaatattcaaagattatctgaaagtccgagattctagattcccgcctgagcgggaatgacgaaaagtggcgggaa tgacggttagcgttgcctcgccttagctcaaigagaacgattctctaaggtgctgaagcaccaagtgaatcggttccgta ctatttgtactgtctgcggcttcgtcgccttgtcctgatttttgttaatccactatctcctgccgcaggggcgggttttg catccgcccgttccgaaagaaaccgcgtgtgcgttttttgccgtctttataacccccggtttgcaatgccctccaatacc ctcccgagtaagtgttgtaaaaatgcaaatcttaaaaaatttaiaataaccaitatgttataiaaacaiaaaaaitaicccataiat atctctatccgtccttcaaaatgcacaitcgaattccacacaaaaacaggcagaagtttgttttiitcagacaggaa Catct atagtttcagacatgtaatcgccgagcccctcggcggtaaatgcaaagctaagcggcttggaaagcccggcctgcttaaa tttcttaaccaaaaaaggaatacagcaatgaaaaaatccctgattgccctgacttitggcagcccttcctgttigcagcaat ggctgacgttaccctgtacggcaccatcaaaaccggcgta SEQ. ID NO: 26 Neisseria meningitidis (serum B group) PorB promoter region

GTTTTCTGTTTTTGAGGGAATGACGGGATGTAGGTTCGTAAGAATGACGGGATATAGGTTTCCGTGCGGATGGATTCGTC

ATTCCCGCGCAGGCGGGAATCTAGAACGTGGAATCTAAGAAACCGTTTTATCCGATAAGTTTTCCGTGCGGACAAGTTTG

GATTCCCGCCTGCGCGGGAATGACGGGATTTTAGGTTTCTAATTTTGGTTTTCTGTTTTTGAGGGAATGACGGGATGTAG

GTTCGTAGGAATGACGGGATATAGGTTTCCGTGCGGATGGATTCGTCATTCCCGCGCAGGCGGGAATCCAGACCTTAGAA

CAAGAGCAATATTCAAAGATTATCTGAAAGTCCGAGATTCTAGATTCCCGCCTGAGCGGGAATGACGAAAAGTGGCGGGA

ATGACGGTTAGCGTTGCCTCGCCTTAGCTCAAAGAGAACGATTCTCTAAGGTGCTGAAGCACTAAGTGAATCGGTTCCGT

ACTATTTGTACTGTCTGCGGCTTCGTCGCCTTGTCCTGATTTTTGTTAATCCACTAT SEQ. ID NO:27 Nuclear chymase sequence (1000 bp) in the DNA region upstream of the siaABC gene in Neisseria meningitidis (serogroup B)

ATACGGCCAATGGCTTCAGAAAGCGATAAGCCTCTGGCTGAAAAACCGATTTCTTGTGTTCTCCCCACCGCACCCATAGA

CGTAAAGGTATAGGGATTGGTAATCATGGTAACCACATCACCGCGACGCAGCAAAATATTTTGTCGCGGATTTGCAACTA

AATCTTCCAAGGCAACAGTTCGTACTACATTGCCACGTGTCAGCTGCACATTCGTATCCTGCACATTTGCCGTTGAACCA

CCTACCGCAGCCACCGCATCCAACACACGCTCACCGGCTGCCGTCAGCGGCATACGCACACTATTCCCAGCACGAATCAC

CGACACATTCGCCGCATTATTCTGCACCAAACGCACCATCACTTGTGGCTGATTGGCCATTTTTTTCAGGCGGCCTTTAA

TAATTTCCTGAACCTGACCAGGCGTTTTACCGACCACCGAAATATCGCCAACAAACGGCACAGAAACCGTACCACGTGCC

GTGACCAACTGCTCTGGCAACTTAGTTTGATGCGCACTACCCGAGCCCATCGAAGAAAGGCCACCACCAAACAATACTGC

CGGCGGCGCTTCCCAAATCATAATATCCAATACATCACCAATATTTAGCGTACCAGCCGAAGCATAACCATCGCCAAACT

GAGTGAATGACTGATTTATCTGAGCCTTATATAATAACTGAGCAACCGTATGATTCACATCAATCAGCTCCACTTCAGGA

ATTTGAACTTCAGATTGTTGCCCTAAAGAGACAATTTTTTTTGCGCTGGGGCCTGATGAAGGAATCGCAGAGCATCCTAC

AATTAAACTTCCACACAATAATAATACTGCGTGACGAATATAAAATTTCACTTTAAACACAAGCCAAATCCTAATATAAT

TATAAATGGCCTAATTATAGCACTTAATCGAAATAAATTTATGAGTACGTAGAGTATAATTAGTATTCTTCTTTCCAACT

TCCTTATACTTATATATATATACTTATAGATTCTAAAATC SEQ. ID NO:28 -100- The paper size applies to the Chinese National Standard (CNS) A4 specification (210 x 297 mm) LIIK.------------r---book-- ------- (Please read the phonetic on the back? Please fill out this page again) 1297731

A7_ B7 _ V. invention is described in (98) Neisseria meningitidis (serogroup B), the nucleotide sequence of the DNA region upstream of the lgt gene (1000 bp) GCCAAAGCATTGGGCGCGGATGCCGCCGCTGCCGAACGCGCCGCGGGTCTTGCCAAAGCCGACTTGGTAACCGAAATGGT CGGCGAGTTCCCCGAACTGCAAGGCACGATGGGCAAATACTATGCCTGTTTGGACGGCGAAACCGAAGAAATTGCCGAAG CCGTCGAGCAGCACTATCAGCCGCGTTTTGCCGGCGACAAGCTGCCCGAAAGCAAAATTGCCGCCGCCGTGGCACTGGCC GACAAACTAGAAACCTTGGTCGGCATTTGGGGCATCGGTCTGATTCCGACCGGCGACAAAGACCCCTACGCCCTGCGCCG CGCTGCCTTGGGTATTTTGCGTATGCTGATGCAGTATGGTTTGGACGTGAACGAACTGATTCAGACGGCATTCGACAGCT TCCCCAAAGGTTTGCTCAACGAAAAAACGCCGTCTGAAACCGCCGACTTTATGCAGGCGCGCCTTGCCGTGTTGCTGCAA AACGATTATCCGCAAGACATCGTTGCCGCCGTACTCGCCAAACAGCCGCGCCGTTTGGACGATTTGACCGCCAAACTGCA GGCCGTTGCCGCGTTCAAAGAACTGCCCGAAGCCGCCGCGCTCGCCGCCGCCAACAAACGCGTGCAAAACCTGCTGAAAA AAGCCGATGCCGAGTTGGGCGCGGTTAACGAAAGCCTGTTGCAACAGGACGAAGAAAAAGCCCTCTTTGCCGCCGCGCAA GGCTTGCAGCCGAAAATCGCCGCCGCCGTCGCCGAAGGCAATTTCCAAACCGCCTTGTCCGAACTGGCTTCCGTCAAACC GCAAGTCGATGCATTCTTTGACGGCGTGATGGTAATGGCGGAAGATGCCGCCGTAAA . ACAAAACCGCCTGAACCTGCTGA ACCGCTTGGCAGAGCAAATGAACGCGGTAGCCGACATCGCGCTTTTGGGCGAGTAACCGTTGTACAGTCCAAATGCCGTC TGAAGCCTTCAGACGGCATCGTGCCTATCGGGAGAATAAASEQ ID NO: 29 Neisseria meningitidis (the MC58 strain), the nucleic acid sequence Chang (1000 bp) DNA region upstream of the gene TbpB GAACGAACCGCSATTCCCACTTTCGTGGGAATGACGAATTTCAGGTTACTGTTTTTGGTTTTCTGTTTTTGTGAAAATAAT GGGATTTCAGCTTGTGGGTATTTACCGGAAAAAACAGAAACCGCTCCGCCGTCATTCCCGCGCAGGCGGGAATCTAGGTC TGTCGGTGCGGAAACTTATCGGATAAAACGGTTTCTTGAGATTTTTCGTCCTGGATTCCCACTTTCGTGGGAATGACGCG AACAGAAACCGCTCCGCCGTCATTCCCGCGCAGGCGGGAATCTAGACATTCAATGCTAAGGCAATTTATCGGGAATGACT GAAACTCAAAAAACTGGATTCCCACTTTCGTGGGAATGACGTGGTGCAGGTTTCCGTATGGATGGATTCGTCATTCCCGC GCAGGCGGGAATCTAGACCTTCAATACTAAGGCAATTTATCGGAAATGACTGAAACTCGAAAAACTGGATTCCCACTTTT GTGGGAATGACGCGATTAGAGTTTCAAAATTTATTCTAAATAGCTGAAACTCAACACACTGGATTCCCGCCTGCGCGGGA ATGACGAAGTGGAAGTTACCCGAAACTTAAAACAAGCGAAACCGAACGAACTGGATTCCCACTTTCGTGGGAATGACGGA ATGTAGGTTCGTGGGAATGACGGCGGAGCGGTTTCTGCTTTTTCCAATAAATGACCCCAACTTAAAATCCCGTCATTCCC GCGCAGGCGGGA . ATCTAGGTCTGTCGGTGCGGAAACTTATGGGGTAAAACGGTTTCTTGAGATTTTGCGTCCTGGATTCC CACTTTCGTGGGAATGACGGAATGTAGGTTCGTGGGAATGACGGGATATAGGTTTCCGTGCGGACGCGTTCGGATTCATG ACTGCGCGGGAATGACGGGATTTTGGTGTATTCCCTAAAAAAATAAAAAAGTATTTGCAAATTTGTTAAAAATAAATAAA ATAATAATCCTTATCATTCTTTAATTGAATTGGATTTATT SEQ ID NO: 30 Neisseria meningitidis (serum group A), the nucleic acid sequence of aluminum (1〇〇〇 bp) DNA region upstream of the gene of opc CAAAGGCTACGACAGT 'GCAACCGTCCGCTGTCG- GCGGAAAA (GGAAACGC;

.CCGGCAACATCTGGAAGAACATCAGTTGTTGGACGGCATTATGCGCAAAGCCTGCC CAAACCAAACGCAACCGGTATTTGTCGAAGACCCGTTATAGTGGATTAAATTTAAAT CAGGACAAGGCGACGAAGCCGCAGACAGTACAAATAGTACGGCAAGGCGAGGCAACGCCGTACTGGTTTAAATTTAATCC AGTATATGTGGTCGAACAGAGCTTCGGTACGCTGCACCGTAAATTCCGCTATGCGCGGGCAGCCTATTTCGGACTGATTA AAGTGAGTGCGCAAAGCCATCTGAAGGCGATGTGTTTGAACCTGTTGAAAGCCGCCAACAAGCTAAGTGCGCCCGCTGCC GCCTAAAAGGAGACCGGATGCCTGATTATCGGGTATCCGGGGAGGGTTAAGGGGGTATTTGGGTAAAATTAGGAGGTATT 3AAAACCTGTG

TGGGGCGAAAATAGACGAAAACCTGTGTTTGGGTTTCGGCTGTCGGGAGGGAAAGGAATTTTGCAAAGATCTCATCCTGT ACAAAAACAGAAAACCAAAAACAGCAACCTGAAATTCGT GAATCTATCGGAAATAACCGAAACCGGACGAACCTAGAT ATAAATGC rAAAAAACAGAAAACCAAGTGAGAATAACAATTCGTTGTAAACA AATATATGTAAAATCCCCCCCCCCCCCCCCCAGAAATCTTAAGAATATAATTGTAAG ACCATATCCGACTACAATCCAAATTTTGGAGATTTTAACT

GCGGCAGGA

AGATTCCCGCTTTCGCGGGAATGACGGCAGAGTGGTTT

CAGTTGCTCCCGATAAATGCCGCCATCTCAAGTCTCGTCATTCCCTTAAAACAGAAAACCGAAATCAGAAACCTAAAATT TCGTCATTCCCATAAAAAACAGAAAACCAAGTGAGAATAACAATTCGTTGTAAACAAATAACTATTTGTTAATTTTTATT AGCGTAACGATTATTTACGTTATGTT BU! ^-------------r---订--------- (Please read the notes on the back and fill out this page) Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative system

SEQ ID NO:. 31 Neisseria meningitidis (serogroup B), the nucleic acid sequence Chang (1000 bp) DNA region upstream of the siaD gene ATAATGCAGGCGCTGAAGTTGTTAAACATCAAACACACATCGTTGAAGACGAAATGTCTGATGAGGCCAAACAAGTCATT CCAGGCAATGCAGATGTCTCTATTTATGAAATTATGGAACGTTGCGCCCTGAATGAAGAAGATGAGATTAAATTAAAAGA ATACGTAGAGAGTAAGGGTATGATTTTTATCAGTACTCCTTTCTCTCGTGCAGCTGCTTTACGATTACAACGTATGGATA TTCCAGCATATAAAATCGGCTCTGGCGAATGTAATAACTACCCATTAATTAAACTGGTGGCCTCTTTTGGTAAGCCTATT ATTCTCTCTACCGGCATGAATTCTATTGAAAGCATCAAAAAGTCGGTAGAAATTATTCGAGAAGCAGGGGTACCTTATGC TTTGCTTCACTGTACCAACATCTACCCAACCCCTTACGAAGATGTTCGATTGGGTGGTATGAACGATTTATCTGAAGCCT TTCCAGACGCAATCATTGGCCTGTCTGACCATACCTTAGATAACTATGCTTGCTTAGGAGCAGTAGCTTTAGGCGGTTCG ATTTTAGAGCGTCACTTTACTGACCGCATGGATCGCCCAGGTCCGGATATTGTATGCTCTATGAATCCGGATACTTTTAA AGAGCTCAAGCAAGGCGCTCATGCTTTAAAATTGGCACGCGGCGGCAAAAAAGACACGATTATCGCGGGAGAAAAGCCAA CTAAAGATTTCGCCTTTGCATCTGTCGTAGCAGATAAAGACATTAAAAAAGGAGAACTGTTGTCCGGAGATAACCTATGG GTTAAACGCCCAGGCAATGGAGACTTCAGCGTCAACGAATATGAAACATTATTTGGTAAGGTCGCTGCTTGCAA TATTCG CAAAGGTGCTCAAATCAAAAAAACTGATATTGAATAATGCTTATTAACTTAGTTACTTTATTAACAGAGGATTGGCTATT ACATATAGCTAATTCTCATTAATTTTTAAGAGATACAATA SEQ. ID NO: 32 Nuclear ruthenium sequence of the upstream DNA region of the ctrA gene in N. meningitidis (serum B group) (1000 bp) ATACCTGCACTTGAGTTGCCGACCATAAATTTAGCATGTTTCAATAAGACTAAAAAATATTCAAATCGAATGGAAGGAAA -101- This paper scale applies to China National Standard (CNS) A4 Specifications (210 x 297 mm) 1297731 A7 B7

GAAACTTCCGCCCCAGCT GGGAAACCGAAAAACGCfl AAGTTTGTCAATCCCACC V. Description of invention (99)

TGCAATAAATTTATCAGATTGATATTTTAATAATTCTTGCAGAATACTTTCAGTGCCAGTGTCATTATTAGGGTAGATGC

TAATGATATTTTGGCCACTTAATTCTAATGCTTTGAAATATTGGGCCGCATATTGTGGCATTAAATGTGCTTCTGTAGTC

ACGGGGTGAAACATAGAAATACCATAATTTTCGTATGGTAAACCGTAATATTCTTTGACTTCTTCTAAGGATGGGAGGGT

GGAAGAGGCCATAACATCTAAATCGGGGGAGCCGATGATGTGAATATGCTTTCTTTTTTCTCCCATTTGCACTAGGCGAG

TGACAGCTTGTTCATTTGCTACCAAGTGGATATGAGAAAGTTTACTAATAGAATGACGAATGGAGTCATCTACTGTACCA

GATAGTTCACCACCTTCGATATGGCAAACTAAACGGCTGCTTAATGCACCTACAGCTGCGCCTGCTAGTGCTTCTAAACG

GTCGCCGTGAATCATGACCATATCAGGTTCAATTTCATCAGATAGACGAGAGATAAACGTAATGGTATTGCCTAAAACGG

CACCCATTGGTTCACCTTGGATTTGATTTGAAAACAGATATGTATGTTGATAGTTTTCTCGAGTTACTTCCTTGTAGGTT

CTGCCATATGTTTTCATCATATGCATACCAGTTACAATCAAATGCAATTCAAGGTCTGGGTGATTTTCAATATAGGCTAA

TAAAGGTTTTAGCTTGCCGAAGTCGGCTCTGGTACCTGTAATGCAAAGAATTCTTTTCATGATTTTAGAATCTATAAGTA

TATATATATAAGTATAAGGAAGTTGGAAAGAAGAATACTAATTATACTCTACGTACTCATAAATTTATTTCGATTAAGTG

CTATAATTAGGCCATTTATAATTATATTAGGATTTGGCTT SEQ. ID NO: 33 Nucleic acid sequence of the DNA region upstream of the lgtF gene in N. meningitidis (serum A population) (1000 bp)

TCTTTTTCGGACTGAAAGGACGCATCATCCCGACATCGAGCGCGTGTTCGTCCGGCAGCCAAGGCATAGGTTATGCCTAC GAAGCCATCAAATACGGTCTGACCGATATGATGCTGGCGGGCGGAGGCGAAGAATTTTTCCCGTCCGAAGTGTATGTTTT CGACTCGCTTTATGCCGCCAGCCGCCGGAACGGCGAACCGGAAAAAACCCCGCGCCCATACGACGCGAACCGCGACGGGC TGGTCATCGGCGAAGGCGCGGGGATTTTCGTGCTGGAAGAATTGGAACACGCCAAACGGCGCGGTGCGATAATTTACGCC GAACTCGTCGGCTACGGAGCCAACAGCGATGCCTACCATATTTCCACGCCCCGCCCCGACGCGCAAGGCGCAATCCTTGC CTTTCAGACGGCATTGCAACACGCAGACCTTGCGCCCGAAGACATCGGCTGGATTAATCTGCACGGCACCGGGACGCACC AGAACGACAGTATGGAAAGCCGCGCCGTTGCAGCGGTTTTCGGCAACAATACGCCCTGCACGTCCACCAAGCCGCAAACC GGACACACGCTGGGCGCGGCGGGCGCAATCGAAGCCGCGTTCGGGTGGGGCATTGCTGACCGGAAAAGCAATCCCGAAGG: TTCCGCCCCAGCTTTGGGACGGGCAGAACGATCCCGACCTTCCCGCCATCAACCTGACCGGCAGC3GCAGCCGCT 3ATTGCCGCCAGCTCGTCGTTTGCCTTCGGAGGAAGCAACTGCGTTTTACTCATCGGATGAAAT: CGCTATGCTATACAATACGCGCCTACTCTTGATGGGTCTGTAGCTCAGGGGTTAGAGCAGGGG ACTCATAATCCCTTGGTCGTGGGTTCGAGCCCCACCGGACCCACCAATTCCCAAGCCCGGACGTATGTTTGGGCTTTTTT GCCGCCCTGTGAAACCAAAATGCTTTGAGAAACCTTGATA SEQ.E) NO: 34 meninges Neisseria meningitidis (serogroup B), the nucleic acid sequence confused (1000 bp) DNA region upstream of the gene lgtB

TAGAAAAATATTTCGCCCAATCATTAGCCGCCGTCGTGAATCAGACTTGGCGCAACTTGGAGATTTTGATTGTCGATGAC

GGCTCGACAGACGGTACGCTTGCCATTGCCAAGGATTTTCAAAAGCGGGACAGCCGTATCAAAATCCTTGCACAAGCTCA

AAATTCCGGCCTGATTCCCTCTTTAAACATCGGGCTGGACGAATTGGCAAAGTCAGGAATGGGGGAATATATTGCACGCA

CCGATGCCGACGATATTGCCGCCCCCGACTGGATTGAGAAAATCGTGGGCGAGATGGAAAAACACCGCAGCATCATCGCG

ATGGGCGCGTGGCTGGAAGTTTTGTCGGAAGAAAAGGACGGCAACCGGCTGGCGCGGCATCACAGGCACGGCAAAATTTG

GAAAAAGCCGACCCGGCACGAAGATATTGCCGACTTTTTCCCTTTCGGCAACCCCATACACAACAACACGATGATTATGA

GGCGCAGCGTCATTGACGGCGGTTTGCGTTACAACACCGAGCGGGATTGGGCGGAAGATTACCAATTTTGGTACGATGTC

AGCAAATTGGGCAGGCTGGCTTATTATCCCGAAGCCTTGGTCAAATACCGCCTTCACGCCAATCAGGTTTCATCCAAATA

CAGCATCCGCCAACACGAAATCGCGCAAGGCATCCAAAAAACCGCCAGAAACGATTTTTTGCAGTCTATGGGTTTTAAAA

CCCGGTTCGACAGCCTTGAATACCGCCAAATAAAAGCAGTAGCGTATGAATTGCTGGAGAAACATTTGCCGGAAGAAGAT

TTTGAACGCGCCCGCGTGTTTTTGTACCAATGCTTCAAACGGACGGACACGCTGCCCGCCGGCGCGTGGCTGGATTTTGC

GGCAGACGGCAGGATGCGGCGGCTGTTTACCTTGAGGCAATACTTCGGCATTTTGCACCGATTGCTGAAAAACCGTTGAA

AAACGCCGCTTTATCCAACAGACAAAAAACAGGATAAATT SEO. ID NO: 35 The nucleotide sequence of the DNA region upstream of the 1st gene in Neisseria meningitidis (serum B group) (1〇〇〇 bp)

GCGCACGGCTTTTTCTTCATCGGTTTGAGGGTCGGCAGGATAATCGGGGACGGCAAAGCCTTTAGACTGCAATTCTTTAA

TCGCGGCGGTCAGTTGAGGTACGGATGCGCTGATGTTCGGCAGTTTGATTACGTTTGCATCGGGCTGTTTCACCAGTTCG

CCCAATTCGGCAAGCGCGTCGGGTACGCGCTGCGCTTCGGTCAGATATTCGGGGAATGCCGCCAAAATACGGCCGGACAG

GGAAATGTCGGCAGTTTTGACATCAATATCGGCGTGGCGGGCAAACGCCTGCACAATCGGCAGCAGCGATTGGGTCGCCA

GCGCGGGGGCTTCGTCGGTATGGGTATAAACAATGGTGGATTTTTGAGTCATAGGATTATTCTCTTGTAGGTTGGTTTTT

TCTTTTGGAACACATTGCGCGGGGAATGTGCGCGGCTATTATGGCATATTTTGGCGGCTTTGTTCGCGCTTTGTTCGATC

TTGGCGTGTTTGAACGCGGCAGCGTGAAAGGAAGGGGGAAAtGGTTTTCCCGCGTTTGGCGGCGGTGTCGGAGGTGCTGT

GCCTGATGTGCGGCGGCATATTTTCGGTGAAATTGATTTTATAGTGGTTTAAATTTAAACCAGTACAGCGTTGCCTCGCC

TTGTCGTACTATCTGTACTGTCTGCGGCTTCGTTGCCTTGTCCTGATTTAAATTTAAACCACTATAATATTCGGTAACTG

TCGGAATATCTGCTAAAATTCCGCATTTTTCCGCCTCGGGACACTCGGGGCGTATGTTTAATTTGTCGGAATGGAGTTTT

AGGGAT SEQ. ID NO: 36 Nucleic acid sequence of the DNA region upstream of the msbB gene in Neisseria meningitidis (serum B group) (1000 bp)

GCCCGACGGCGAACAGACACGTCGTGAAATCAACCGCTTGGACAGTAGGGCGGCGCAATACGACATGCTTGCAGGTTATC rGCCGGAAAAACCGACCGTTGGGCGTGCGCCTACCGCCAAAATGCCGTCTGAACACCCGATTATCCTTTT rTATGCCCCATACCCTTCCCGATATTTCCCAATGTATCAGACAAAATTTGGAACAATATTTCAAAGACCT GAACGGTACCGAACCTTGCGGCGTGTACGATATGGTCTTGCATCAGGTGGAAAAACCGCTGCTGGTGTGCGTGATGGAAC AATGCGGCGGCAACCAGTCCAAAGCCTCCGTCATGTTGGGACTGAACCGCAATACTTTGCGTAAAAAACTGATTCAACAC GGTTTGCTGTGAATATGTCGGCAACCGTCCGTATCTTGGGTATTGACCCGGGCAGTCGCGTAACGGGTTTCGGTGTCATC GATGTCAGGGGGCGCGATCATTTTTACGTCGCCTCCGGCTGCATCAAAACGCCTGCCGATGCGCCTCTGGCAGACAGGAT TGCCGTGATTGTGCGGCATATCGGCGAAGTCGTTACCGTTTACAAGCCTCAACAGGCGGCAGTGGAACAGGTGTTCGTCA -102- Table paper scale applicable Chinese National Standard (CNS) A4 size (210 x 297 mm) ------------ ----- r-- -Order--------- (Please read the notes on the back and fill out this page.) Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed TTGAAAi GAAAGCi A7 1297731 V. Invention Description (100)

ACGTCAATCCGGCATCGACGCTGATGCTCGGTCAGGCTAGGGGCGCGGCATTGGCGGCATTGGTCAGCCATAAGCTGCCC

GTTTCGGAATACACGGCCTTGCAGGTCAAACAGGCGGTAGTCGGCAAGGGCAAGGCGGCAAAAGAACAGGTGCAGCATAT

GGTGGTGCAGATGCTGGGGCTTTCGGGAACGCCGCAGGANTGGCGGCGGACGGTCTTGCCGTCGCGCTGACCCACGCCTT

ACGCAACCACGGGCTTGCCGCCAAACTCAATCCTTCGGGGATGCAGGTCAAGCGCGGCAGGTTTCAATAGTTTCAGACGG

CATTTGTATTTTGCCGTCTGAAAAGAAAATGTGTATCGAG SEQ.ID NO:37 Nuclear citrate sequence of the upstream DNA region of htrB gene in N. meningitidis (serogroup B) (1000 bp)

CCGCCAAGCGTTTCCCCCTTTGTCGGGCTTAACATTTGCTTTGTACGGCAGACTTTTTCCCTTCATAACGCCGCCTTTCC

GAAAAGACGATGGTAGGCGCGACGTAATTCTCAACCCTTAAGGTACGGTTGGACGAAAAGTTTTCCTTTTbATTCCACCT

GCCAACTTTTCGGCTACACCGAGTGGTCTCGTTAGGTTTGGGCGAACTACGCCCTTAAAAAAACGGACATTCTTTGCATG

CCCGTCTCTAAGGTTTCACGGTAAGTTTACCCTTATAAAGAGTTGACTTACCATACTTATCCCTTTAAAACGATATAAAG

GGCGACAGCTGTAATACAAGTATGTTGTACGGCAGACTTCTTCTACCAAACAAAAAGTTCCTTTTAGAGTTACTCGCTTA

TAGACAAATGAAGGCTTAGCCATAGGCTTCCGGTAGGCCTATTTCAACGGCTGGTTCACAGGCTACGCTAAAACCTACGG

TAGAACCGCGTTCTGGGGTTTCGCGCACAGCGGCGTCTTTGGAACCAGTTGTGTCCGAACACGCATAACCGCCCGCTTTA

ATGGTGGTGGCGGGTTCACCTGATGTAGTTTCAGCGTGCGCTTTGGTAGTTTGCGTAGCCGATGTTGAGGAGGCTCGACC

CGAAACTACGGTTGCCGACGCGCCAGCCGCACATGATGCTGGTCGTTAGAGGCCTGTAGCGGGTTCCGCACTTGCTTCCG

CTTCCGTAACTGAACTTGGTTCCGCGACCGCTGGTTCCAAACTACAAGCCGATACGGACGCTGCTTTGGGGCTGGGACTA

CGGCAAACGGTAGATAATGTCGGTGGCGGACTACGTCGCAGTTTCGCTTAATGCGTTTCTGCCGGAGGACGGAACCGACG

CAGGGCTGCGTTTTCGGGTTGACTGGCACCAAATGCTATCGCTTAGGCCGTTTCATTTTGCGTAACTATGGCAGCAGGAG

AGATACGTTGTGCTGGGCCTTTAGCCAATACTTCTCAACT SEQ.IDNO: 38 The nucleoside sequence of the DNA region upstream of the MltA gene in N. meningitidis (serogroup B) (1000 bp)

TGTTTGAA AACGCCAT

CGCCC

CACAAAAACCAAGTTATGACGGGAATAAGGTACAGCAGCCAAACCAAGGCCTCGCCCTGC GTCGGATGGTCGGTATAGGCGAAAAATCCGCCGAGCAGCACGCCCAACGGGCTGTCTTCG TGCAAATATTTTGATGAGTCGAACACAATGTCCTGAAGCGCGTTCCAAATGCCTGCTTCG TGCAGCGCACGCAGCGAACCGGCAAGCAGACCAGCGGCAACGATAATCAGAAACGCCCCT GTCCAACGGAAAAACTTCGCCAGATTCAGGCGCATCCCACCCrGATAAATCAACGCGCCA ATCACGGCGGCAGCCAAAACCCCCGCrACCGCACCGGCCGGCATCTGCCACGTCGGGCTC GAATACGGCAAGCAGGAAAAAAACGCTCTCCAAACCTTCGCGCGCCACGGCAAGA ATACCGACCAAGGCCCATCCTTGACCGCTGCCACGGTTCAAAGCCGCCTGCACA GAATCCTGAAGCrGCCGCTTCATCGAACGGGCGGCTTnTrCATCCATAAAATCATATAA GTCAGCATCGCGACAGCAACCAAACCGATAATGCCGACGACGAACrCCTGCTGCTTCTGG GGAATCTCGCCCGTTGCCGAATGGATTCCGTACCCCAGCCCCAAACACATCAAAGAAGCA AGAACAACCCCGAACCAGACCTTAGGCATCAGTTTGGAATGTCCGGACTGTTTCAGAAAA CCGGCAACGATGCCGACGATGAGCGCGGCTTCGATACCCrCGCGCAACATAATTAAAAAA GCGACCAGCATAAACGCGAACGAACAAGGATGATGAATAATATATTATCGGAATATTTTC ATTGCrrGTAAATACAAATGCAAGTrATTrTTATCTGCAGTACCGCGCGGCGGAAAGTTC CGCAGCTGCAGCTGCGCCCrGTGTTAAAATCCCCrcrCCACGGCTGCCGCAACGCCG GAAACCATCTrrCTTATrACTGCCGGCAACATrGTCCAT T SEQ. ID NO: 39 The nucleotide sequence of the upstream DNA region of the ompCD gene in Moraxella catarrhalis (1000 bp)

GCTGATTTGTGAGCAAGCGGGCGCATCAGGGATTACCTTGCATTTGCGAGAAGATCGTCG

ACATATTCAAGATGAAGATGTTTATGAATTGATTGGGCAATTGACAACACGCATGAATCT

TGAGATGGCAGTCACTGATGAGATGCTAAATATTGCCCTAAAGGTACGACCAGCATGGGT

GTGTTTAGTACCAGAAAAACGCCAAGAGCTGACTACAGAAGGTGGGCTTGATATCGCCAA

TTTATCAAATATTCAAGCATTTATACACAGTCTTCAGCAGGCGGATATTAAGGTTTCTTT

ATTCATCGATCCAGATCCGCATCAAATTGATGCTGCAATTGCTTTGGGTGCTGATGCGAT

TGAGCTGCATACGGGAGCTTATGCTCAAGCGACTTTACAAAATAATCAAAAGCTTGTTGA

TAAAGAGCTTGACCGTATTCAAAAAGCCGTTGCAATGGCACAAAAAAAATCATCATTATT

GATTAATGCAGGTCATGGTTTGACGCGTGATAATGTTGCAGCGATTGCCCAAATTGATGG

TATTCATGAGCTGAATATCGGGCATGCATTGATTTCAGATGCGATATTTATGGGGCTTGA

TAATGCAGTCAAGGCAATGAAAATGGCTTTTATTCAAGATAAAACGACCAATCATTGATG

CGTTAGAAAGAAAATCGTAAATAATGATGACTATTGTGTAATATTATGTATTTTTGTTCA

AAAAAAGGTTGTAAAAAAATTCATTTACCATTAAGCTAAGCCCACAAGCCACAATGAATA

CCTATTGGTTTGACTCATTAGTCACTAAGAATCTGCAAAATTTTGTAACAGATTATTGGC

AGGTCTTGGATCGCTATGCTAAAATAGGTGCGGTAATCTTGAAAAACCAACCATTCCTTG

GAGGAATTTATGAAAAAGGGATATAAACGCTCTTGCGGTCATCGCAGCCGTTGCAGCTCC

AGTTGCAGCTCCAGTTGCTGCTCAAGCTGGTGTGACAGTC DNA upstream region of copB gene SEQ. ID NO: 40 Nucleic acid sequence in Moraxella catarrhalis (1000 bp)

GATGCTGTTAAAGTGGGTATTGGTCCTGGTTCTATTTGTACAACCCGTATTGTTGCAGGC ATTGGCGTCCCGCAGATAAGTGCCATTGATAGTGTGGCAAGTGCGTTAAAAGATCGCATT -103- 1 This paper scale applies to China National Standard (CNS) A4 specification (210 297 297 mm Γ 1297731 A7 B7 V. Invention Description (101)

-GCCAAAG 3GAAGAAG

GGCGCTTCATGTATTATGGTGGGTAGCTTGTTGGCAGGTACCGAAGAAGCACCTGGTGAG GTGGAATTATTCCAAGGTCGTTATTATAAGGCTTATCGTGGTATGGGCAGCTTGGGGGCA ATGTCTGGTCAAAATGGCTCATCGGATCGTTATTTTCAAGATGCCAAAGATGGTGTTGAA AAACTGGTTCCAGAGGGTATCGAAGGCCGTGTTCCTTATAAAGGCCCTGTGGCAGGCATC ATCGGTCAATTGGCAGGTGGTCTAAGATCATCCATGGGTTATACAGGTTGCCAGACCATC GAACAGATGCGTAAGAATACCAGCTTTGTCAAAGTGACTTCCGCAGGCATGAAGGAATCG CATGTACACGATGTACAGATTACCAAAGAAGCACCCAATTATCGCCAAAATTAACTCTAT TAATAGCAAATACAAGCACTCATTAGATAGGGTGGGTGCTTTTTAGAGCATA ACTGACACATGACTTATTGTCATATTTTTAAAATGCTTTTAATTTAGATTTTTft ATAATGGCTAAAAATAACAGAATATTAATTTAAAGTTTTCAAAATCAAGCGATTAGATGA AATTATGAAAATAAATAACAATAATTCTGATTTATTTTAACCAATAATATCAATTATCAT TTACAAGAAAAATTTTTTTTGATAAAATTCTTACTTGTACCTTGCTATTTTTTCTTATTT ATCATTTTTGGCGGTATTTTCGTTGATTTTAGTAAGTAGATGAGCAAGGGATAATTTGAC AAAAACAAATTTGATTTCAAGCCTCATAATCGGAGTTATT

ΓΑΑΑΑΑΑΤΑΑ rTTAATTTAG

SEQ ID NO:. 41, k cards it would Laplace, the nucleic acid sequence confused (1000 bp) DNA region upstream of the gene DI5 AAAACTGGTGATGTCTTCACTGCTATTCATGGTGAACCAATCAATGATTGGCTAAGTGCC ACCAAGATTATTCAGGCAAATCCAGAAACCATGCTTGATGTGACAGTCATGCGTCAAGGT AAGCAGGTTGATTTAAAATTAATGCCCCGTGGTGTAAAGACACAAAACGGCGTAGTCGGT CAACTGGGTATTCGCCCCCAGATTGATATCGATACGCTCATTCCTGATGAATATCGTATG acgattcaatatgatstcggtgaggcatttactcaagggatcggacgaacttatgattta TCAATAATGACCTTAGATGCGATGGGTAAGATGATTACAGGATTGATTGGCATTGAAAAT CTATCAGGTCCCATTGCCATTGCCGATGTTTCTAAGACCAGTTTTGAGTTGGGATTTCAA GAAGTGTTATCGACAGCCGCAATCATCAGTTTAAGCTTGGCAGTACTGAATCTTTTACCC ATTCCAGTGTTAGATGGCGGGCATTTGGTATTTTATACTTATGAATGGATtATGGGCAAA TCTATGAATGAAGCGGTGCAGATGGCAGCATTTAAAGCGGGTGCGTTATTGCTTTTTTGT TTCATGTTACTTGCAATCAGTAACGATATCATGCGATTTTTTGGCTAAGTTCTGATTTAT CGTACCATTAACAAAATTTTTGGCTTTTTTAAGCTGAAATAGTTGCCAAATTTAACTTTT TGGCTTACCTTTACACAATATAAATTTGGGTGTAGAAAATTTTGGATACATTTTTATACC TTATTTTTAGAAATTTTAAAAATTAAGTTTGGATAGACTTATGCGTAATTCATATTTTAA AGGTTTTCAGGTCAGTGCAATGACAATGGCTGTCATGATGGTAATGTCAACTCA TGCACA AGCGGCGGATTTTATGGCAAATGACATTGCCATCACAGGACTACAGCGAGTGACCATTGA AAGCTTACAAAGCGTGCTGCCGTTTCGCTTGGGTCAAGTG Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing

SE〇 ID NO:. 42 Moraxella catarrhal, the nuclear DNA area upstream of the gene for omplA acid sequence (1000 bp) ACTTGGCGAAAATACCATTTATATCGATTGTGATGTTATACAGGCAGATGGCGGTACACG CACAGCCAGTATCAGTGGTGCTGCGGTGGCACTTATTGATGCTTTAGAACACTTGCAGCG TCGTAAAAAGCTTACCCAAGATCCGCTTTTGGGCTTGGTGGCAGCGGTTTCTGTGGGTGT TAATCAAGGCCGTGTATTGCTTGATTTGGATTATGCTGAAGATTCAACTTGTGATACCGA TTTAAATGTGGTCATGACGCAGGCAGGTGGGTTTATTGAGATTCAAGGCACAGCAGAAGA AAAGCCATTTACTCGTGCTGAAGCTAATGCGATGCTTGATTTGGCAGAGCTGGGAATTGG GCAGATTATCGAAGCCCAAAAGCAAGTATTAGGCTGGTGATATGCTAATCGTTGAAGATA ATGGCGTGATCATCACATTAAATGGACAAGTAAAAGACCCATTATTTTGGTGGTCGATGA TATTGCTGCTGCTGGGTGTCTTGGTGGCAATCATTTGTTTGATTGCACCCGTTTTTTATG CAATCGGTGCGTTGGCTTTATTTGCAGTTGTGGTATTTGTGTTTAATATTCAAAGGCAAA AAGCCAAAACTTGTCATATGTTTTCACAAGGTCGCTTGAAGATTACGTCCAAACGCTTTG AGATTCATAACAAATCACTAACCTTATCAGCATCGGCAACAATATCTGCTAAAGATAACA AAATGACAATTGTTGATCGGGGCATTGAATATCATTTTACAGGTTTTGCTGATGACCGTG AAATTAATATAGCCAAACAGGTACTTTTGGGAAAGTCAATCAAAACCAATGCGGTGGCGG TAACATTGGCTAAGTAGTTGTTGTGATACAGACAGGTTGGATGGTCTTTA ACTCCACCCA cctaactttttctttgtttggatttaagagtatgttatgatgggcaggattttattttaa GTCATCATTTAATGCAATCAGTTGTCCAGAGTAGCCGTTC

SEQ ID NO:. 43 Moraxella coli, the nucleic acid sequence taste (1000 bp) DNA region upstream of the gene hly3 GTGATCGGCAACACCCCACCATTCAGGAGCAACCAAAATTGCCCGTGCCTTGCCTGTCTT GGTGGTATCATTTGGCAGGGCAATGTGGCTAAGTAGTGGTGTGCCATCAGGTGCGGTGGT GGTGAGTGTACGATTCGTTATTGTCATAAAATTATCCTTTTGGGTTGGATGATATCAATG AAATACCCTACGGTTGTATGGAATTTTATCCATTGTACCACGGTATTGGTCTTTTTAAAT TAACAAGCAGCTTCTAGCAAGTCAAAGTTTTTATGCCTATTTTTTCAGATTTTAAGGTAC AATAAAGCCAATTGTTAATAATATGGTATTGTCATGATTTATGATGAATTGCGACCAAAA TTTTGGGAAAATTATCCCTTAGATGCGTTAACAGATGCTGAATGGGAAGCATTATGTGAC GGATGTGGCGCGTGTTGTTTGGTGAAATTTCTTGATGATGACAATGTTAAATTGACCGAA TATACCGATGTTGCCTGCCAGCTATTGGATTGCTCAACAGGATTTTGCCAAAACTATGCC AAGCGTCAAACGATTGTGCCAGATTGTATTCGCTTAACACCTGATATGCTGCCTGATATG CTGTGGTTGCCACGCCATTGTGCTTATAAGCGGTTGTATCTTGGGCAAAATCTGCCAGCA TGGCACAGGCTCATTAAACATAGCCAAAACCATGGTGCAGGATTTGCGAAAGTTTCAACT GCTGGGCGATGTGTGAGTGAGCTTGGTATGAGTGATGAAGACATAGAAAGGCGAGTGGTG -104- This paper applies the Chinese national scale Standard (CNS) A4 specification (210 x 297 mm) ----^-------------r--I -------- (Please read the notes on the back and fill out this page) 1297731 A7 B7 Description of invention (102) AAATGGG'; GTTAAA( 'AAAATT&lt;

CCTTGACATGATTGTTGACATGATTGACAGACAATAAAAATTGGCAAA CCGAGTGA

ΓΑΑΑΑΑ' TTTGATAAAATTGGTGTATGTGTGTGATTTTATCAAAAGCACTTGAATAAAAi TACGCTAAATTGTAGCAAACCAATCAATTCATCATAATTTTAATGAACACGAGGTTAAAT TATACTGTCTATGTCTGATGACAATTCAAGCACTTGGTCG

. SEQ ID NO: 44 Moraxella coli, the nucleic acid sequence Chang (1000 bp) DNA region upstream of the gene lpbA TAACAAAGGCAACCCAACACGCAGTTATTTTGTGCAAGGCGGTCAAGCGGATGTCAGTAC TCAGCTGCCCAGTGCAGGTAAATTCACCTATAATGGTCTTTGGGCAGGCTACCTGACCCA GAAAAAAGACAAAGGTTATAGCAAAGATGAGGATACCATCAAGCAAAAAGGTCTTAAAGA TTATATATTGACCAAAGACTTTATCCCACAAGATGACGATGACGATGACGATGACGATAG TTTGACCGCATCTGATGATTCACAAGATGATAATACACATGGCGATGATGATTTGATTGC ATCTGATGATTCACAAGATGATGACGCAGATGGCGATGACGATTCAGATGATTTGGGTGA TGGTGCAGATGATGACGCCGCAGGCAAAGTGTATCATGCAGGTAATATTCGCCCTGAATT .CAAATACTTGCCCATTAATGAGCCTACTCATGAAAAAACCTTTGCCCTAGATGG TGAAAA (TAAAAAf

3AAAATTG SGGTAACA

TACCGCCAAAGCCGATGTGCCAAACTATCGTGAAGAAGTGGGTAACAACCAAGGTGGCGG TTTCTTATACAACATCAAAGATATTGATGTTAAGGGGCAATTTTTTGGCACAAATGGCGA AGAGTTGGCAGGACGGTTACATCATGACAAAGGCGATGGCATCACTGACACCGCCGAAAA AGCAGGGGCTGTCTTTGGGGCTGTTAAAGATAAATAAAGCCCCCCTCATCATCGTTTAGT CGCTTGACCGACAGTTGATGACGCCCTTGGCAATGTCTTAAAACAGCACTTTGAAACAGT GCCTTGGGCGAATTCTTGGATAAATGCACCAGATTTGCCTCGGG ATAAAGTTTAGGATTTTTTT

3GCTAATATCTTGATAAA

. ACATCGCCATAAAATAGAAAAT SEQ ID NO: 45 Moraxella catarrhal in the nuclear Chang acid sequence (1000 bp) CAGCTTGTACCATTTGGTGAATATATACCATTTGGTGGTTTGTTGGATATTTTACCAGGG CTTGAGGGTGTCGCTAGCCTAAGCCGTGGCGATGATAAGCAACCACCGCTCAAATTGGGC GGCGGCGTGGGCGATACGATTGGTGCGGCAATTTGTTATGAGGTGGCATATCCTGAGACG ACGCGTAAAAATGCACTTGGCAGTAATTTTTTATTAACCGTCTCAAACGATGCTTGGTTT GGTACAACAGCAGGTCCTTTGCAGCATTTACAAATGGTGCAAATGCGAAGCTTGGAGACG GGGCGATGGTTTGTGCGTGCAACAAACAACSGAGTGACTGCATTAATTGACCATCAAGGA CGGATTATCAAGCAGATACCGCAGTTTCAGCGAGATATTTTGCGAGGTGATGTACCCAGT TATGTTGGACACACGCCTTATATGGTTTGGGGGCATTATCCCATGTTGGGGTTTTCTTTG GTGCTGATTTTTCTTAGTATCATGGCAAAGAAAATGAAAAATACCACCGCCAAACGAGAA AAATTTTATACCGCTGATGGTGTGGTAGACCGCTGAATTGTGCCACTTTGGGCGTTAGAG CATGAGCAAGATTAGGCGTTGGGTGAGCTTTGGTTGTATTACTCATCAGCCTACCCGAAA CCTGCCAAACATCACCGCCCAAAACCTAAACATACAATGGCTAAAAATATCAGAAAATAA CTTGCTGTATTGTAAATTCTTATGTTATCATGTGATAATAATTATCATTAGTACCAAGAT ATCCATTACTAAACTTCATCCCCCATCTTAACAGTTACCAAGCGGTGAGCGGATTATCCG ATTGACAGCAAGCTTAGCATGATGGCATCGGCTGATTGTCTTTTTGCCTTGTTGTGT GTT TGTGGGAGTTGATTGTACTTACCTTAGTGGTGGATGCTTGGGCTGATTTAATTAAATTTG ATCAAAGCGGTCTTCACAACACACCAAACGAGATATCACC lbpB gene upstream DNA region μ — 祖一--------ΦΜ ——— l·---订· ------- (Please read the back of the note before you fill out this page ) _ SEQ. ID NO: 46 Nucleic acid sequence in Moraxella catarrhalis (1000 bp) tbpB gene upstream DNA region Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing

AGTTTGCCCTGATTTTGAGAGCCACTGCCATCATGAATTTGTTGGCGTAAACACCACTCG TATTCTTCTTCGGTTTCCCCTTTCCATGCAAACACAGGGATACCAGCGGCCGCCATGGCA GCGGCGGCGTGGTCTTGGGTGCTAAAAATATTGCATGATGTCCAGCGAACTTCTGCACCC AAGGCAACCAAAGTCTCAATCAGCACCGCTGTTTGAATGGTCATGTGGATACAGCCTAGG ATTTTAGCACCCTTAAGTGGTTGCTGGTCTTGATAGCGTTTTCTTAACCCCATCAGGGCT GGCATCTCAGCTTCTGCCAAGGCAATCTCACGGCGACCATAATCGGCTAAACGGATATCA GCGACTTTATAATCGGTGAAGTTTTGGGTGGTACTTGGATTGATTGAGGTAGGCATATCT TTATTCCTAAGCTATTTTAAAGTATTTTTAACAATAATTTTGATGAATTTGAGATAATTG ATGCTAAAAGGTTGAATGACCAAACCATCGCTAACAATCAAGAAAAGACATTTTAAGCAT AAAAAGCAAATGTGTCTTGATGGCTTATTATAACAGTTATTATGATAAATTTGGGTAGAA AGTTAAATGGATCGTTGGGTAAGTTTGTTGGCTATCCTTAATTAATTATAATTTTTTAAT AATGCTTTTACTTTATTTTAAAAATAGAGTAAAAAATGGTTGGCTTTGGGTTTTTATCTC ACTATGGTAGATAAAATTGATACAAAATGGTTTGTATTATCACTTGTATTTGTATTATAA TTTTACTTATTTTTACAAACTATACACTAAAATCAAAAATTAATCACTTTGGTTGGGTGG TTTTAGCAAGCAAATGGTTATTTTGGTAAACAATTAAGTTCTTAAAAACGATACACGCTC ATAAACAGATGGTTTTTGGCATCTGCAATTTGATGCCTGCCTTGTGATTGGTTGGGGTGT ATCGGTGTATCAAAGTGCAAAAGC CAACAGGTGG7CATTG SEQ. ID NO:47 -105- The paper size applies to the Chinese National Standard (CNS) A4 specification (210 x 297 mm) 1297731 A7 B7

V. Description of the Invention (1〇3) Moraxella catarrhal, the nuclear DNA area upstream of the tbpA gene for acid sequence (1000 bp) TTGGGGGCGGATAAAAAGTGGTCTTTGCCCAAAGGGGCATATGTGGGAGCGAACACCCAA ATCTATGGCAAACATCATCAAAATCACAAAAAATACAACGACCATTGGGGCAGACTGGGG GCAAATTTGGGCTTTGCTGATGCCAAAAAAGACCTTAGCATTGAGACCTATGGTGAAAAA AGATTTTATGGGCATGAGCGTTATACCGACACCATCGGCATACGCATGTCGGTTGATTAT AGAATCAACCCAAAATTTCAAAGCCTAAACGCCATAGACATATCACGCCTAACCAACCAT CGGACGCCCAGGGCTGACAGTAATAACACTTTATACAGCACATCATTGATTTATTACCCA AATGCCACACGCTATTATCTTTTGGGGGCAGACTTTTATGATGAAAAAGTGCCACAAGAC CCATCTGACAGCTATGAGCGTCGTGGCATACGCACAGCGTGGGGGCAAGAATGGGCGGGT GGTCTTTCAAGCCGTGCCCAAATCAGCATCAACAAACGCCATTACCAAGGGGCAAACCTA ACCAGTGGCGGACAAATTCGCCATGATAAACAGATGCAAGCGTCTTTATCGCTTTGGCAC AGAGACATTCACAAATGGGGCATCACGCCACGGCTGACCATCAGTACAAACATCAATAAA AGCAATGACATCAAGGCAAATTATCACAAAAATCAAATGTTTGTTGAGTTTAGTCGCATT TTTTGATGGGATAAGCACGCCCTACTTTTGTTTTTGTAAAAAAATGTGCCATCATAGACA ATATCAAGAAAAAATCAAGAAAAAAAGATTACAAATTTAATGATAATTGTTATTGTTTAT GTTATTATTTATCAATGTAAATTTGCCGTATTTTGTCC . ATCACAAACGCATTTATCATCA ATGCCCAGACAAATACGCCAAATGCACATTGTCAACATGCCAAAATAGGCATTAACAGAC TTTTTTAGATAATACCATCAACCCATCAGAGGATTATTTT SEQ ID NO: 48 Moraxella catarrhal Chang acid sequence of nuclear DNA region upstream of the Ministry of Economy ompE gene Intellectual Property Office employee of consumer cooperative printed (1000 bp) AAAGACATTACACATCATCATTCAAACGCCCAACCATGTACCTCTGCCCCGTGGTCGCAC GCCAACGCTTTTTGATGCGGTGCGTTGGGTTCAGATGGCTTGTCAATCATTTGGTTTTAT TAAAATTCATACCTTTGGTAGTTTGGCTTTACCTGATATGTCATTTGATTATCGAAACAA TACGCAGTTGACCAAACATCAATTTTTAGCCATTTGCCAAGCACTCAATATTACCGCTCA TACGACCATGCTTGGTATTAAATCATCACATAAAGATACTTTACATCCATTTGAATTGAC ATTACCCAAATACGGCCATGCCTCAMTTATGATGATGAATTGGTGCAAAACAATCCATT GGCTTATTTTCATCAACTGTCTGCCGTCTGCCGATATTTTTATACCCAAACGGTTTGTAT tgttggcggtgaaagctcagggaaaactaccttggtgcaaaaacttgccaattattatgg TGCCAGCATCGCACCTGAAATGGGTCGATTATACACACACTCCCATCTCGGCGGTAGCGA ACTTGCCCTTCAATACAGCGACTACGCATCCATTGCCATCAATCACGCCAACGCTATCGA AACCGCTCGTACCACTGCCAGCTCTGCTGTTACACTGATTGATACTGATTTTGCGACAAC GCAAGCATTTTGTGAAATTTATGAAGGGCGAACGCATCCGCTTGTCGCAGAATTTGCTAA . ACAAATGCGATTGGATTTTACGATTTATTTAGATAATAATGTTGCTTGGGTCGCTGATGG CATGCGTAGGCTTGGTGATGATCATCAACGCAGTTTGTTCGCCAATAAATTGCTTGAGAT TTTGGCACGATATGATATTAGTTATCATATCATTAATGACACCGACTACCACAAACGCTA TCTACAAGCATTAAGCTTGATAGACAATCATATTTTTAATCATTTTACAAAAATTCATGA CAATTAATTAGGGAAAATCTGATGAAAATTGATATTTTAG SEQ ID NO: 49 Moraxella catarrhal in the nuclear Chang acid sequence (1000 bp) GGATGTGGCATATCTGCCCATCGACCCAATACACATCGGTCGAGGCTATCAAGATGTGGT ACGAATTAATAGCCAGTCAGGTAAGGGCGGTGCTGCGTATATCTTGCAGCGGCATTTTGG TTTTAATTTACCACGCTGGACACAGATTGATTTTGCTCGTGTGGTACAGGCTTATGCAGA AAGTATGGCGCGTGAACTAAAAACTGATGAGCTGCTTGAAATTTTTACCCAAGCGTATCT TAAGCAAGATAAATTCCGCCTAAGTGACTATACCATCAGCAATAAAGGCGATGCTGTCAG CTTCCAAGGCCAAGTAGCGACACCCAAAGCGGTGTTTGAGGTGATTGGTCAAGGCAATGG TGCGTTATCTGCGTTCATTGATGGCTTGGTGAAATCCACAGGCAGACAGATTCATGTCAC CAATTACGCCGAACACGCCATCGATAACAAAACCCATCAAAAAACCGATACGGATAACCA AACCGATGCCGCCGTGCCGCTTATATCCAGCTGTCGGTAGAGGGGCAGATTTATTCAGGC ATCGCCACTTGCCATAGCACCGTATCCGCCATGCTAAAAGGTGCATTATCCGCTTTGGCA CAGGCGTGGTAATCTGACCCAATCAAAATCCTGCATGAT . GGCAGGATTTTATTATTTAGT GGGCTGCCCAACAATGATGATCATCAGCATGTGAGCAAATGACTGGCGTAAATGACTGAT GAGTGTCTATTTAATGAAAGATATCAATATATAAAAGTTGACTATAGCGATGCAATACAG TAAAATTTGTTACGGCTAAACATAACGACGGTCCAAGATGGCGGATATCGCCATTTACCA ACCTGATAATCAGTTTGATAGCCATTAGCGATGGCATCAAGTTGTGTTGTTGTATTGTCA TATAAACGGTAAATTTGGTTTGGTGGATGCCCCATCTGATTTACCGTCCCCCTAATAAGT GAGGGGGGGGGAGACCCCAGTCATTTATTAGGAGACTAAG SEQ ID NO: 50 Moraxella coli, the nucleic acid sequence Chang (1000 bp) DNA region upstream of the gene uspa2 CCCCAAGCTTTCCGTTTGTGTGCCTGCTGGTGTCGGGCGGTCATACCATGCTGGTGCGTG CCGATGGTGTGGGCGTGTATCAGATATTGGGCGAGTCTATCGATGATGCGGTGGGTGAAT GCTTTGATAAAACGGCAAAAATGCTCAAACTGCCCTATCCTGGTGGCCCAAATATCGAAA AATTAGCCAAAAACGGCAACCCACACGCCTATGAGCTGCCAAGACCCATGCAGCATAAAG GGCTGGATTTTTCGTTCAGTGGCATGAAAACCGCCATTCATAATCTCATCAAAGACACAC CAAACGCCCAAAGCGACCCCGCCACACGAGCAGACATCGCCGCAAGCTTTGAGTATGCGG TGGTGGATACTTTGGTCAAAAAATGCACCAAAGCACTACAGATGACAGGCATTCGCCAGC 106- uspal DNA region upstream of the gene of the present The paper scale applies to the Chinese National Standard (CNS) A4 specification (210 x 297 mm) (Please read the note on the back first? Matters to fill out this page) Order -------! 0, 1297731 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (104)

TGGTGGTCGCAGGGGGCGTCTCTGCCAATCAGATGCTACGCCGCACCCTGACCGAGACGC

TCCGCCAAATCGATGCGTCGGTGTACTATGCCCCGACCGAGCTATGCACGGATAATGGTG

CGATGATCGCCTATGCTGGCTTTTGTCGGCTCAGCTGTGGACAGTCGGATGACTTGGCGG

TTCGCTGTATTCCCCGATGGGATATGACGACGCTTGGCGTATCGGCTCATAGATAGCCAC

ATCAATCATACCAACCAAATCGTACAAACGGTTGATACATGCCAAAAATACCATATTGAA

AGTAGGGTTTGGGTATTATTTATGTAACTTATATCTAATTTGGTGTTGATACTTTGATAA

AGCCTTGCTATACTGTAACCTAAATGGATATGATAGAGATTTTTCCATTTATGCCAGCAA

AAGAGATAGATAGATAGATAGATAGATAGAACTGTGTCTTTTATCTGTCCGCTGATGCTT

TCTGCCTGCCACCGATGATATCATTTATCTGCTTTTTAGGCATCAGTTATTTCACCGTGA

TGACTGATGTGATGACTTAACCACCAAAAGAGAGTGCTAA SEQ. IDNO: 51 Nucleic acid sequence of the upstream DNA region of the 〇mp2 1 gene in Moraxella catarrhalis (1000 bp)

GAGTGAACTTTATTGTAAAATATGATTCATTAAAGTATCAAAATCATCAAACGCAGCATC

AGGGTTTGCTAAATCAATTTTTTCACCATAATTATAGCCATAACGCACAGCAAGCGTAGT

TATGCCAGCGGCTTGCCCTGATAAAATATCATTTTTGGAATCACCAACCATAATGGCATC

AGTCGGTGCGATGCCCAGTGATTGACACAGGTATAATAAAGGCGTTGGGTCGGGCTTTTT

GACGCTGAGCGTATCACCGCCAATCACTTGGTCAAACAGTGTCAGCCATCCAAAATGTGA

TAAAATTTTAGGCAAATAACGCTCAGGCTTATTGGTACAAATTGCCAAATAAAACCCCGC

TGCTTTTAATCGTTCAAGCCCTTGTATAACCCCTGCATAGCTTTGCGTATTTTCAATTGT

TTTATGGGCATATTCTGCCAAAAATAACTCATGGGCATGGTGAATCATAGTCGTATCATA

GATATGATGTGCTTGCATTGCTCGCTCAACCAATTTTAGCGAACCATTGCCCACCCAGCT

TTTGATGATATCAATTGGCATAGGCGGTAAGTTAAGCTTGGCATACATGCCATTGACCGC

CGCCGCCAAATCAGGGGCACTATCGATAAGGGTACCATCCAAATCAAATATAATCAGTTT

TTTGCCAGTCATTGACAGTGTTTGGATGCTTTTTCCTTATTCTTAAAATTGGCGGCTGTT

TGGTATTTTTTAAATCAGTCAATTTTTACCATTTGTCATATAATGACAAAGTACAAATTT

AGCAATATTTTAGTGCATTTTTTGGCGAAGTTTTATGAAAACTGGTCATTGGTTGCAAAA

CTTTACACAGTACCTATAAAACTTGCACAGTTAATAAGAAATATTTTGTTACTATAGGGG

CGTCATTTGGAACAAGACAGTTATTTGTAAATAGTTATTTGCAAAAGACGGCTAAAAGAC

AGAACAGCGTTTGTTTCAGTGATTAACTAGGAGAAAAACA SEQ. ID NO: 52 In the Moraxella catarrhalis, the nucleotide region of the upstream DNA region of the 〇mpl06 gene (1000 bp) TTGATCGGTTTTGCCCCACTGTTTCATGATTTACTCAAAACAGGCGGCTTGATCGTGCTG* GCAGGTCTGACCCAAAACCAAACCCAAGCGGTCATCGATGCCTACTCGCCTTATGTTACC?

CTTGATACGCCATTTTGTTATGCAGATGCCCAAGACTGCCATTGGCAACGCCTAAGCGGC

ATCAAACCTACCAACCCATAAGCGATATGCCATGAGCCACAAACCTAAGCCAACACCGCT

ATATCAACAAGTTGAGCAGACCGCCAAGCGTTATTTTGAGACATTGGGCGATGCTCATAC

TCATGATGTCTATGCCACTTTTTTGGCCGAATTTGAAAAACCGCTGCTCATCGCCGCACT

CAATCACACGCACGGCAATCAGTCAAAAACCGCCCAAATCCXTGGTATCAATCGTGGCAC

ATTACGCACCAAAATGAAAACCCATCACTTACTTTAGACCGCCAGTTATCGCCATGGATA

TGGGCAGGTGTGCTCGCCTGCCGTATGATGGCGATGACACCCCATTTGCCCCATATCTGC

ACGATTTGACATGATTTAACATGTGATATGATTTAACATGTGACATGATTTAACATTGTT

TAATACTGTTGCCATCATTACCATAATTTAGTAACGCATTTGTAAAAATCATTGCCCCCT

TTTTTTATGTGTATCATATGAATAGAATATTATGATTGTATCTGATTATTGTATCAGAAT

GGTGATGCCTACGAGTTGATTTGGGTTAATCACTCTATTATTTGATATGTTTTGAAACTA

ATCTATTGACTTAAATCACCATATGGTTATAATTTAGCATAATGGTAGGCTTTTTGTAAA

AATCACATCGCAATATTGTTCTACTGTTACCACCATGCTTGAATGACGATCCAAATCACC

AGATTCATTCAAGTGATGTGTTTGTATACGCACCATTTACCCTAATTATTTCAATCAAAT

GCCTATGTCAGCATGTATCATTTTTTTAAGGTAAACCACC SEQ. ID NO: 53 ^ Nucleic acid sequence of the upstream DNA region of the HtrB gene in Moraxella catarrhalis (1000 bp)

ACTATTCTGCTTTTTGTTTTTCACGAATGCGAATGCCCAACTCACGCAACTGGCGATTAT

CAACTTCAGCAGGTGCTTCGGTCAATGGGCAATCTGCCGTCTTGGTTTTTGGGAAGGCGA

TCACATCACGGATTGAGCTGGCACCAACCATCAGCATAATCAGGCGATCTAGACCAAATG

CCAAACCACCGTGCGGCGGTGCACCAAAACGCAATGCATCCATCAAAAACTTAAACTTAA

GCTCTGCTTCTTCTTTAGAAATACCCAAGGCATCAAATACCGCCTCTTGCATGTCAACCG

TATTAATACGCAGCGAACCGCCACCAATTTCTGTGCCATTTAGTACCATGTCATAGGCAA

TGGATAGGGCGGTTTCGGGACTTTGTTTGAGTTCCTCAACCGAGCCTTTTGGGCGTGTAA

AAGGATGATGAACTGATGTCCACTTACCATCATCAGTTTCCTCAAACATTGGAAAATCAA

CGACCCAAAGCGGTGCCCATTCACAGGTAAATAAATTTAAATCAGTACCGATTTTAACAC

GCAATGCACCCATAGCATCATTGACGATTTTGGCTTTATCGGCACCAAAGAAAATGATAT

CGCCAGTTTGGGCATCGGTACGCTCAATCAGCTCAATCAAAACCTCATCGGTCATATTTT

TAATGATGGGTGATTGTAATCCTGATTCTTTTTCAACGCCATTATTGATATTGCTTGCGT

CATTGACCTTAATATATGCCAATCCACGAGCGCCATAAATACCAACAAATTTGGTGTACT

CATCAATCTGCTTGCGACTCATGTTACCGCCATTTGGAATGCGTAAGGCAACAACACGGC

CTTTAGGATCTTGGGCGGGCCCTGAAAATACTTTAAATTCAACATGTTGCATGATGTCAG

CAACATCAATAAGTTTTAAGGGAATGCGTAAATCAGGCTTATCTGAGGCATAATCACGCA -107- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) l·----------^^--------- (please read first On the back of the phonetic 2, please fill out this page) 1297731 A7 B7 V. Description of invention (105)

TGGCATCTGCGTAAGTCATGCGGGGGAAGGTATCAAACTCA SEQ ID NO:. 54 Moraxella bacteria's' core Chang acid sequence (1000 bp) DNA region upstream of the MsbB gene TGGATCATATTCTTTATTAATGGTACTGTTTAAACCTGTATTTTAAAGTTTATTGGGTCA TATTTTCAAGCTCATCCCATCGCTCAAGCTTCATCATCAAAAGCTCATCAATCTCTACCA ATCGCTCACCA-GCCTTCGTTGCTGCCGCCAAATCGGTATTAAACCATGAACCATCTTCAA TCTTTTTGGCAAGCTGTGCCTGCTCTTGTTCAAGTGCAGCAATTTCATTAGGCAAATCTT CAAGTTCACGCTGCTCTTTATAGCTGAGTTTGCGTTTTTGGGCAACGCCTGATTGAGGTG GTTTGATTTGGATGGGTTCAGCGGGTTTTGTCGCCTTAGGTTTATTGTCTGTGGCGTGAT GAGCAAGGCATCTTTCATGCTGTTGTACATAGTCTTCATAACCGCCAACATATTCCAAAA CGATACCGTCGCCGTACTTATCAGTATCAAATACCCAAGTTTGGGTAACAACATTATCCA TAAAAGCACGGTCATGGCTGATGAGTAATACCGTGCCTTTAAAATTGACCACAAAATCTT CTAAAAGCTCAAGTGTTGCCATATCCAAATCATTGGTAGGCTCATCAAGCACCAAAACAT TGGCAGGTTTTAGCAATAATTTGGCCAATAAAACGCGTGCTTTTTCACCGCCTGATAGTG CTTTAACAGGTGTGCGAGCACGATTTGGCGTGAATAAAAAATCTTGCAAATAGCTTAAAA TGTGCGTAGTTTTTCCACCAACATCGACATGGTCAGAGCCTTCTGAAACATTATCTGCGA TAGATTTTTCAGGGTCTAGGTCGTCTTTGAGTTGGTCAAAAAAAGCAATATTTAGATTGG TGCCAAGC . TTAACTGAACCTGACTGAATCGCTGAATCATCCAAACCCAAAATGCTTTTAA TTAAGGTTGTTTTACCAACGCCATTTTTGCCAATGATACCAACTTTATCACCACGAACAA GCAGCGTTGAAAAATCCTTAACTAAGGTTTTATTGTCGTAT SEQ ID NO: 55 Moraxella coli, the nucleic acid sequence Chang (1000 bp) DNA region upstream of the PilQ gene CAACTTGAAAATCAGCTCAATGCTCTGCCACGCACAGCACCGATGAGCGAGATTATCGGA ATGATAAATACCAAAGCACAAGCGGTTAATGTGCAGGTGGTGAGTGCATCAGTTCAAGCA GGTCGTGAACAGGATTATTATACCGAACGCCCTATCGCAGTGAGTGCGACAGGGGATTAT CATGCTTTGGGTCGATGGTTACTTGAGTTGTCAGAGGCTAACCATTTGCTGACAGTGCAT GATTTTGATCTGAAGGCTGGTTTGAACCATCAGCTGATGATGATTGTTCAGATGAAAACT TATCAAGCGAACAAACGCCCAAAACCAGTTGCTCAGCAGGTGCCTGATGTTCAATGAATA TTATCGGTGGGGCATTTTGGGTGCTTGGATTTGGGTTGGGATTGGATGTGCTGATAGCAC CAGTCAAGTTGTTGATGATAAGCTTGCACATATTACCGATGAAGAGCGTATGGCGATCAG TGAGCCTGTGCCGATACCCTTATCTGTGCCGATGATATATCAGCAAGGCAAAGATCCTTT TATCAATCCTTATAGAAATGTTGAGGTTCTTGATACCAATCATGCCGCTGATCAGCAAGA TGAGCCAAAAACCGAATCTACCAAAGCTTGGCCTATGGCAGACACTATGCCATCTCAGCC ATCTGATACTCATCAGTCTGCCAAGGCTCAGGCACAAGTCTTCAAAGGCGATCCGATAGT CATTGATACCAACCGTGT TCGAGAGCCTTTAGAAAGCTATGAGTTATCAAGCCTACGCTA TCATGGTCGTATTTTTGATGATGTTAGACTTGTGGCACTCATTATGAGTCCTGATGGCAT CGTTCATCGTGTGAGTACTGGACAATATCTTGGTAAAAATCACGGAAAAATTACCCATAT TGACAGTCGTACGATACATCTGATTGAAGCGGTCGCTGATACACAAGGTGGCTATTATCG ccgtgatgtaaacattcattttattcataagcaatgacac SEQ ID NO:. 56 Moraxella coli, the nucleic acid sequence plan (1000 bp) DNA region upstream of the gene lip〇18 (Read Notes on the back and then fill the page) the tube set ---------Dance · Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing

TTCATGCAACAAGCGACCATCTTGGCCGATGATACCATCCTGCTCACCTAAGAAAATCAG TTTATCAGCTTGCAGGGCAATGGCTGTGGTCAGTGCTACATCTTCTGCCAATAGATTAAA AATTTCGCCCGTAACCGAAAAACCTGTCGGTCCTAGTAGGACAATATGGTCATTATCCAA ATTATGGCGAATGGCATCGACATCAATTGAGCGTACCTCACCTGTCATCTGATAATCCAT ACCATCTCTGATGCCGTAAGGGCGAGCGGTGACAAAATTACCCGAAATGGCATCAATACG AGATCCGTACATTGGGGAGTTAGCAAGCCCCATCGACAGCCGAGCTTCGATTTGTAGACG AATTGAGCCGACTGCCTCCAAGATGGCAGGCATAGATTCATACGGTGTTACACGCACATT CTCATGTAGGTTTGATATCAGCTTGCGATTTTGTAAATTTTTTTCCACTTGTGGGCGTAC ACCATGCACAAGCACCAATTTGATGCCCAAGCTGTGTAGCAGTGCAAAATCATGAATCAG CGTACTAAAATTGTCACGAGCGACCGCCTCATCACCAAACATAACCACAAAGGTTTTGCC ACGATGGGTGTTAATGTACGGGGCAGAATTACGAAACCAATGCACAGGTGTGAGTGCAGG AGTGTTCTGATAGGTGCTGACAGAATTCATGAATGCTCCAAAGAGTCAATGGCTGGTAAA ATAAGAATGGCGAACAATATATGGCGAGAGCGTCTGATGTTGGTCAAATGTCCCATTAAT AACTATCAAGATACCATCATACCATAGCAAAGTTTTGGGCAGATGCCAAGCGAATTTATC AGCTTGATAAGGTTGGCATATGATAAAATCTACCATCATCGTCGCCAGTTTTGAGCATGT GTAAGTAGTTACCATAATTAAACAGTCAAGAAATTCACACCGTCAATCAGCTGTGCTATG TAAAACT CTTATGGGCACAT ^

fVCTTGACCAACACAGGATAAATTTA SEQ ID NO:. 57 Moraxella catarrhal, the nuclear DNA area upstream of the gene for lipoll acid sequence (1000 bp) GGCATACTTTTGCCATGCTTTATTTTGGCATAACTGCTATAAGCCCATTGCTACTTTTTA TCATTTATCCATATGTCCAATAATGTGCTTTATGTAATTTAGGCACACTATTAACTCGTG CCACTGTTAACATTCAGCATAAAAATCTTAACAATGAATCAAAGCATCGTATTGGCTGTT AAATGATAAGCTTATAT-TTATTTAAATTCAGACTAAATGATTGTAATATGGACATATCAA -108- suitable for the present paper China National Standard Scale (CNS) A4 size (210 X 297 mm) 1297731 A7 B7 Description of invention (1〇6)

GGTTGAAATCAAAAATTTTGGAGAGTTATGTACGATAATGATAAAAAATTGACCACCATC GTAGGGGTGTTGTATACGGTGTCTTATATTGCCATATGGTTGGTCAGTGGCTATATTTTA TGGGGCTGGATTGGTGTGACAGGATTTACTCGTGCGATACTTTGGCTGATCGCTTGGATG ATTGTGGGTACGATTGCTGATAGAATTCTGATACCGATTA'iTTTGACCGTCGTGGTTGGG TTATTTTCTATCTTTTTTGAAAAAAGGCGATAATTTGGTTATTTTTTCACAAAAAATGAT GATTTTTTTTGTAAACTATCTAAAATATCAATTATGTTATATTATGTGATAAAAGATGGG CATGCTTAAGTTTTGGATTGCAAAAATCCTAATATCATCACTGACCAAAGCTGTGATGAT ATCAAAACTTTATCAAAGTTCTTAGGGTATTATCAAGATATCATACCAAATGAATACTTA CCCAACTTACTATAAAAATCAAATGATATGACTGTGATTTTATTATCATAGATACAAAAA ACGCATGAGCCAAAGGTATGATGAATGAATACAAAATTTCGCACACATTATGACA ATGTCGCCAGAAACGCTGACATTGCGGTGATTTGGTGGGATAGGGGTCAAGCCA GTGCGATTAAGCTAAATTTTTATGTGGGCAATGGCTGACTTTATTTTATTTGTGCCAGTT GGAACAATTCGTGGTCTAATGTATTTATTTTAAGGAGATAA

TCAAAACG ATCTAAAT SEO ID NO:. 58 ^, A i wide pull it into a card bacteria, nuclear DNA area for 1ip〇10 acid sequence upstream of the gene (1000 bp) TCTGGTCTACATCCCAAACTATTTACACAAGAAACACTAAAGACAGTGGAGGAGATGACG CTCAAAAAGGCATCTTATAGTAATTTGACAGTTAATTTTCGTCAAGTGCTTGTACAAAAA TACACCATCGTGCAAGAAGTTTGTACCAATTTAAGCACAATCATTTTGGCACACACTGTC AAGCAATGCTTCAGGCAAATTAGCTGCTGGTAAAGATACTTGGGTCATCATGCAATCGCA TCAACCCTTCTTGCTGCGTTGAAGCGATAAGTTTGCCATCTTGCCAAAATTGACCATGGT TTAGACCCTTGGCGTGGCTTGTGGTATCGCTCCACATGTCGTAGAGTAGATATTCGGTCA TATCAAAAGGGCGATGGAAATGTATGGAATGGTCAATACTAGCCATTTGTAGACCTTGTG TCATCAGGCTTAGCCCATGACTCATTAAACCTGTGCTGACCAAATAATAATCAGACACAA ACGCAAGTAGTGCTTGATGAATGGCAACTGGCTGCTCCCCAATATCAGCGATACGCACCC AATTGGCTTGGCGTGGACGCTCAGGCTTGGGTGTCACAGGGTCTCGTGGTGTGACGGGGC GGATTTCGACATGACGCTGACGCATAAATCTTGGTTTGAGTGGTTCGGGAATTTTATGTA AATAATCCGCTTTGAGTTCTTGCTCGGTTTTTAGGCTTTCAGGGGGTGGATAATCAGGCA TGGTTTCTTGGTAATCAAGCCCGCCTTCCATGGGTGAAAATGAGGCAATCATCGAAAAAA TGACCTGTTCATTGGTCGTATGATTACCGTTTTTGTCGGTGGTTGGCACATATTGCACCG CAATGACTTCTCGAGCTGAT AAACTGCGTCCATCACGTAAGCGGCGTACTTGATAGATGA CTGGTAGACGAATATCGCCACCTCGTAAAAAATAACCATGTAGGCTATGACAAGGTTTAT CAATCGTTAATGTGTTAGCACCAGCAAGCAGCGCTTGGGCASEQ. ID NO: 59 in Moraxella catarrhalis, the nucleotide region of the upstream DNA region of the lipo2 gene (1000 bp)

.TTATATGTTCAAAAATCGCTTAAGTATTGAAAAAAGC

TAAAATGACCTTACAAAATAAAAT * TATAAAAACTTATCTATTAAAGCATAAAAGATATTAAAGCATAAAAGACGAGAAAAGAGC AAGCGTCAATGATGATATTTCATATAAAAACTTATGAAATTTTTCAATTTTTTATCGATT GATTCAGCTTGGCTATCGGTGGTCAACTTTGGCTGCCAAGACATCGCCGGCTTTTTGAAA AATCATCACAATGGCAACAATGATGATGGTTGAAATCCACTTGACATATACCATGTTGCG ATGCTCACCATAGTTAATCGCAAGGCTTCCCAAGCCACCACCGCCAACCACACCTGCCAT TGCAGAATAACCAATCAAAGACACCAAGGTCAATGTGACCGCATTAATCAAAATGGGCAG GCTTTCAGCAAAATAGTATTTGCTGACTVACCTGCCAATGCGTTGCACCCATAGATTTGGC AGCTTCGGTCAGTCCTGTGGGTACTTCTAATAAAGCATTGGCACTCAAGCGTGCAAAAAA TGGAATTGCTGCCACACTCAAAGGGACGATGGCGGCTGTTGTGCCAAGGGTTGTTCCCAC CAAAAAT AATATTAATAATAACATCCAAAATTACAAATACACTGCGATTTTCAAGGATACGCCCTTT ATGCCAAAAACCCTATCGGTAGCCCAACC AGCAAGCCCCATATAGATGGTTTCCCAAGTGGATTGGGCAACCATCTCCCACATTCTTGG GTGCATTTCACTGACAAATTTTGTGACGATTTCATTCCACATAGCCGATAATCTCAATAT TGACCCGATGGGTGGTTAAAAATTCTATTGCTTGCATGACCGAGGTGCCTTCACCGATAA 3CCAAAT ATCGGTT. TCGTGTGACTGGCATGAGAATAATGAGCAAAATAATAAAAGGAACGGAGCGACC ΤΑΑΤΑΑΤΛ ΤΤΑΑΑΑΑΊ

:CAAAAC CAACCAT l· --.-------------r------------ (Please read the notes on the back and fill out this page)

GCTCAGCAATGGTAAAGC

ATTTTATATCACCTGCATAA economic Intellectual Property Office employee consumer cooperative printed SEQ ID NO:. 60 Moraxella catarrhal, the Ta core Πρ〇7 acid sequence (1000 bp) DNA region upstream of the gene AGTAAACAATGGTAACAAATACAGCAGTGTCGCACAGTCCTCAGTACGATGATTCTGAAT TTGAATATGCAGGATTTTGGATACGATTTGTGGCATGTCTTGTCGATAATTTAATTGTTA TGATTATAATTGCACCGTATTGGTTTTATAATTATCAGCAAATGATGGCCATGCCTGCTG ACCAAATACCGTTTTATAGTGTTGGGGATGCCATCCTTTATAGTGCTGGGGATGCTATCG TAAACTTAGTGATGGCGGCGGCGGTTGTTTGGTTTTGGGTAAAAAAAGGTGCAACACCAG GTAAAATGCTCTTTGGGCTGCAAGTCCGTGATGCCAAAACAGGGCAATTTATCAGTGTGC CAAGGGCATTATTGCGATATTTTAGTTATCTGATTTCATCCGTGATTCTTTGTTTGGGAC TTATTTGGGTTGGTTTTGATAAGAAAAAACAAGGCTGGCATGATA TTGTGGTAAAACGCATTCGCTGATGGGTCGCCAGTTAAACAATfl GCAGGGCGATGTGTTTGAGCAGTTGGCGGTAGATAAGCTAAAACAAGCAGGCTATGAAAT TATTTTAACCAACTTTACCACCCCATTTGTTGGTGAGATTGATATTATCGCCAGACAGCC TTTGGAGCAATCGCACCGTTTGGTGCAGCCAAGATTTTGTACGGTATTTGTTGAAGTGCG TAGCCGAACAAGTTCTGTGTATGGTACAGCGCTTGAGAGTGTTACCTCAAAAAAGCAGGC AAAAATCTACCGAACAGCAGAACGATTTTTAATCAATTATCCCAAATATATTGATGATGC ATACCG TTTTGATGTCATGGTTTTTGATTTGGTTGATGGATTGATTGAACATGAATGGAT AAAAAATGCGTTTTGATTGGCTCAATGGTCGTGAATTAAAATCAATCAAGCAATCCGTAG-109-

ATAAAATTGCCAAAACTG rAAAACCATCAAACGCAA This paper scale applies to China National Standard (CNS) A4 specification (210 x 297 mm) 1297731 A7 B7 V. Invention Description (1〇7) Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing

CTTTACTATAAGATATATCCCAGTAATATGGAAACATAGCASEQ ID N0:. 61 Moraxella coli, the nucleic acid sequence Chang (1000 bp) DNA region upstream of the gene of Προ6 CGTTTAGCTTCATACGCAGACCTTGTGCACCTTCGGGCAACCGAAGCATCACGCCAGCAT CACGCATCCGCACAAAACCCATCATGCCATCAATTTCGCTGCTGATATGATATACCCCCA CCAAAGTAAACCGCTTAAATCGTGGAATAACGCCTGCTGCTGAGGGTGAGGCTTCAGGCA AAACCAAGGTAACCTTATCCCCCAACTTAAGTCCCATGTCAGAGACAATGGACTCACCTA ATATAATACCAAACTCGCCGATATGTAAATCATCCAAATTGCCTGCGGTCATATGCTCAT CAATGATAGAAACTTGCTTTTCGTAATGAGGCTCAATGCCAGAAACCACGATTCCAGTCA CCTGACCTTCAGCGGTTAACATACCTTGTAGTTGAATATAAGGGGCAACTGCTTGCACTT * CTGGATTTTGCATTTTGATTTTTTCGGCAAGTTCTTGCCAATTTGTCAAAATTTCTGTTG AGGTAACTGAAGCTTGAGGCACCATGCCAAGAATGCGTGATTTAATTTCACGGTCAAAGC CATTCATGACCGACAAAACCGTGATAAGCACTGCAACCCCAAGCGTAAGCCCAATGGTTG AGATAAAAGAAATAAAGGAAATAAAGCCATTTTTACGCTTAGCTTTGGTATATCTAAGCC CAATAAATAACGCCAAGGGACGAAACATAAGCTGTGTTGCAAACGACCCAACCGTGCTAG TTTAGCACTTTTTTGGACAAATACCAAACATCACATAACAAATGAATCATCAGGTTGGTT TTGTTGCGCTTGTGTATCTGTATGATAAGTTTCTTGCTAAAACAGCTTTTTTATGTCAGA ATACAGA AAAGGTATATACTTATATTTTTAACTTTAAATAGATCTGCTTTTTTATACCGA TGATTTGGCATGAAGTTTATCGGTCTGATATGCTGGATATAAGTTTATCGGCTTGATATA AATTTTAATTAATCATCAAATTTTTAAGGAATT.TATCATTASEQ ID NO:. 62 · Moraxella catarrhal, the nuclear DNA area upstream of the gene for P6 acid sequence (1000 bp) TAAGGATACCAGATTTTGGCTTGTCAATCGTTGTGTTAATCATTGTAACGGTTTATAGTG ATTGTCAATTAATAAGGGTAAAAAAGTATTTATCAAGTAATAATCTTTCTTATATGTGAA TATAATGACAAATTTATCACATTTTTACAAGGATTTTTTATCAAGATTAGGATATGTTCC AGCTTAATTATTAGTGATGAGCGTGTGATTATTTGGCATCGTTAAATTTATGAGTGCTAA AATTGCCAAATGATTAAAATTTTGCTAACATGATAdCCCCTTTGGTAGGCTTTATTTGGT ATTGATGAGCAATAATAATATACCGAGTTAAATGGATTAACTTAACATACGCCAAAAACT TAACAACGftAAAGTAGATGATTATGACAGATACAGTACAAAAAGATACAGCACAGTCCCC CAAAAAAGTTTATCTAAAAGACTACACGCCGCCAGTATATGCAGTTAATAAAGTGGATTT GGATATCCGCTTGTTTGATGATCATGCTGTCGTTGGTGCCAAACTTAAAATGACACGAGC ACACGCAGGCGAGCTTCGGCTTCTTGGGCGAGATTTAAAGCTTAAAAGCATTCACCTAAA * TGGTCAGGAATTAGAGTCGCAGGCGTATCATCTTGATAAGGAAGGCTTAACAATTTTAGA TGCACCAGATGTCGCAGTGATTGAGACATTGGTTGAGATTTCACCACAAACCAACACAAC ACTTGAAGGGCTAT ATCAAGCAGGAACAGGTGATGATAAGATGTTTGTGACACAATGCGA ACCTGAGGGTTTTCGCAAAATCACCTTTTTCCCTGACCGCCCTGATGTTTTGACAGAATA CACCACACGCCTAGAAGCACCAAAGCATTTTAAAACCTTGCTTGCCAATGGTAATTTGGT TGAGTCAGGAGATGTGGATGAAAATCGCCATTATACCATTTGGCATGATCCTACCAAAAA ACCCAGCTATCTATTCGCCGCTGTCATTGCCAATCTAGAAGSEQ ID NO:. 63 Haemophilus influenzae (HIRD), the core region of DNA upstream of MsbB gene for acid sequence (1000 bp), AAATCAAGCGCCTGTGCCTGCTGGTGATGGTTGTGGAGACGAATTATATTCTTGGTTTGA ACCGCCAAAACCAGGCACTTCAGTGAGCAAACCTAAAGTTACACCGCCTGAGCCGTTTTT GTGCCAACAGATTTTGAACTCACCGAATCGGAGAGAATGGTTAGAATAGCATTGAGGTAA ATCAATATGGATATCGGCATTGATCTTTTAGCAATATTGTTTTGTGTTGGTTTTGTCGCA TCATTTATCGATGCAATTGCTGGCGGTGGTGGATTAATCACCATTCCAGCGTTACTCATG ACAGGTATGCCACCAGCAATGGCGTTAGGCACCAACAAATTGCAAGCTATGGGCGGTGCA TTATCCGCAAGCCTTTATTTCTTGCGAAAAAGAGCGGTCAATTTACGCGATATTTGGTTT ATTTTGATTTGGGTTTTCTTAGGTTCTGCCCTAGGTACATTATTAATTCAATCAATTGAC GTGGCGATTTTCAAAAAAATGCTTCCTTTTTTGATTTTAGCCATTGGTCTATATTTTTTA TTTACTCCTAAATTAGGTGATGAAGATCGAAAACAACGATTAAGTTATCTGTTATTTGGT CTTTTAGTTAGCCCATTTT TAGGTTTTTATGATGGCTTCTTTGGGCCAGGGACTGGCTCA ATCATGAGTTTAGCCTGTGTTACTTTGCTAGGATTTAATCTCCCGAAAGCGGCAGCACAT GCAAAAGTGATGAACTTCACTTCGAACCTTGCTTCTTTTGCACTTTTCTTATTGGGCGGA CAAATTCTTTGGAAAGTGGGTTTCGTGATGATGGCTGGGAGCATTTTAGGTGCAAATTTA GGTGCCAAAATGGTGATGACGAAAGGTAAAACCTTGATTCGACCGATGGTTGTTATCATG TCTTTTATGATGACGGCTAAAATGGTTTACGATCAGGGTTGGTTTCATTTTTAATTCGGA GTTAAAATT AAGCGCGCAAAAGTGCGGTT ^

fVTTAATTACATTTTATTA SEQ ID NO:. 64 Haemophilus influenzae (HIRD) in 5 Chang acid sequence (1000 bp) DNA region upstream of the HtrB gene, TTGAAGTCCCCAATTTACCCACCACAATTCCTGCGGCAACATTGGCTAGGTAACAAGATT CTTCGAAAGAACGTCCATCTGCTAATGTGGTTGCTAATACACTAATGACAGTGTCACCGG CTCCCGTCACATCAAACACTTCTTTTGCAACGGTTGGCAAATGATAAGGCTCTTGATTTG GGCGTAATAATGTCATGCCTTTTTCAGAACGCGTCACCAAAAGTGCGGTTAATTCAATAT CAGAAATTAATTTTAAACCTTTCTTAATAATCTCTTCTTCTGTATTACATTTACCTACAA -110- suitable for the present paper China National Standard Scale (CNS) A4 Specifications (210 X 297 mm) u------------1 —IL----^-------— ^__wi (Please read the notes on the back and fill in the form) Page) 1297731 A7 B7 V. Description of invention (1〇8)

CGGCTTCAAATTCAGACATATTGGGTGTCAATAATGTAGCCCCACGATAACGTTCAAAAT CAGTTCCCTTTGGATCGATCAACACAGGCACATTCGCTTTGCGTGCAATTTGAATCATTT TCTGAACATCTTTAAGCGTGCCTTTGCCGTAATCAGAAAGAATCAAAGCACCGTAATTTT TCACCGCACTTTCTAACTTCGCTAATAAATCCTTGCAATCTACATTATTGAAATCTTCTT CAAAATCAAGGCGGAGCAGCTGTTGATGACGAGATAAAATACGTAATTTAGTAATGGTTG GATGGGTTTCTAATGCAACAAAATTACAATCAATCTTTTGTTTTTCTAATAAGTGGGAAA GT.GCAGAACCTGTCTCATCTTGTCCAATCAATCCCATTAACTGAACGGGTACATTGAGTG AAGCAATATTCATCGCCACATTTGCAGCACCGCCCGCGCGTTCTTCATTTTCTTGTACGC GAACTACTGGCACTGGTGCTTCTGGTGAAATACGGTTGGTTGCACCGAACCAATAACGAT CAAGCATCACATCGCCTAATACAAGTACTTTTGCTTGCTTAAATTCTGCTGAATATTGAG CCATTTTAAAATCTCTCTATTTGAATAACCAAAATTGTGGCGATTTTACCACAACTCAAA TGGAAAG

TTTACGATAAACTACGCCCCTAACTTACGTG

^GAACAA

SEQ · ID N〇: 65 Haemophilus influenzae (HIRD), the nucleic acid sequence Chang (1000 bp) DNA region upstream of the protein D gene AGCAATAATTATAGCTGGAATATTCTTTAAAGATGAAAGAGATCGTATAAGACAAAAAGA ATTTTATATTGGAGAATTATTAGCAATTATTGGTTCGCTAATATTCGTAATAAATAGTTC AAATAATGATGGAAATACAGACTTTTTTCTTGGGGCAATATTTCTTTTTACAGCTATTTT TATTCAATCTGTACAGAATTTAATTGTAAAAAAAGTAGCCAAAAAGATAAATGCTGTTGT AATAAGTGCATCGACAGCAACAATTTCAGGAGTATTATTTTTATGTTTAGCTTTTAATAC TAAACAAATATATTTATTACAAGATGTTGGCATTGGAATGTTGATAGGTTTAGTTTGCGC TGGCTTTTATGGGATGCTAACAGGGATGTTGATGGCTTTTTATATTGTTCAAAAACAGGG AATCACTGTTTTTAACATTTTGCAATTATTAATTCCTCTTTCAACTGCGATAATAGGTTA CTTAACATTAGATGAAAGAATAAATATCTATCAGGGAATTAGCGGTATTATTGTAATTAT TGGTTGTGTATTGGCATTAAAAAGAAAAAACAAGGAGTGTTGATATATAAAGTAGATGAT GTTGGTGGAATAGGTATAGTTAAATATCTGGTTCAATTGGTTTTATTAAGGGCGTTAGCA ATTCTCCATTTAAGTTTATGTTTGAATTAGATATTTTGGGAAAAGATGGAAGAATAAAGC TGTTAAATAATGCTGAAACATATGAACTATACCAATACTCAAATAAAAATAATTCTGCTG .TTATAAATCTCTAATTCTAACTTGTAGAGAGGATAATGACTATCAATCAGAAA TAAAGCCATTAAAAATATTATTCATTGTATGACTAATAATCATCA ACCTATTT : TGAAACATCTTTAGAAACTATTAAAATTATTCACGGAATAATTAATTCTGTTA AAATAGGTAATGATCCTAACAATATATAAGGAGAATAAGT ;AAATGAf ;AATGATTAAAG&lt;CAAGTGCTGAAACATCTTT; G:

SEQ ID NO:. 66 H. influenzae (HiRd), Hin47 core Chang acid sequence (1000 bp) DNA region upstream of the gene TAAATACTCCAAAATAAATTTCAGATAACGTGGTCTGTAAGACAAAAAAATAAAAAAAAT GTTCAATAAGAGGAGAGCAAATTATCTTGTTTAAAAGGAAATCGGAGCAGTACAAAAACG GTCTTACAAGTAGCAAATTCTATAAATTTATGTTCTAATACGCGCAATTTTCTAGTCAAT AAAAAGGTCAAAAAATGAGCTGGATTAACCGAATTTTTAGTAAAAGTCCTTCTTCTTCCA CTCGAAAAGCCAATGTGCCAGAAGGCGTATGGACAAAATGTACTGCTTGTGAACAAGTAC TTTATAGTGAAGAACTCAAACGTAATCTGTATGTTTGCCCGAAATGTGGTCATCATATGC GTATTGATGCTCGTGAGCGTTTATTAAATTTATTGGACGAAGATTCAAGCCAAGAAATTG CGGCAGATTTAGAACCAAAAGATATTTTAAAATTCAAAGATTTAAAGAAATATAAAGATC GTATCAATGCGGCGCAAAAAGAAACGGGCGAGAAAGATGCGCTAATTACTATGACAGGTA CACTTTATAATATGCCAATCGTTGTGGCTGCATCGAACTTTGCTTTTATGGGCGGTTCAA TGGGTTCTGTAGTTGGTGCAAAATTTGTTAAAGCGGCTGAAAAAGCGATGGAAATGAATT GTCCATTTGTGTGTTTCTCTGCGAGTGGTGGTGCTCGTATGCAGGAAGCATTATTCTCTT TAATGCAAATGGCAAAAACTAGTGCCGTACTTGCTCAAATGCGTGAAAAGGGTGTGCCAT TTATTTCAGTATTAACGGATCCGACTTTAGGCGGCGTATCAGCCAGTTTTGCGATGTTAG GGGATTTAAATATTGCCGAGCCAAAAGCCTTAATTGGTTTTGCAGGG CCACGCGTTATTG AACAAACTGTGCGTGAAAAATTGCCAGAAGGTTTCCAACGTAGTGAGTTTCTACTTGAGA AAGGGGCAATTGATATGATCGTGAAACGTTCAGAAATGCGT l·——·----.----装----hi order--------- (please read the notes on the back and fill out this page) Ministry of Economics Intellectual Property Office workers consumer cooperative printed SEQ ID NO:. 67 H. influenzae nuclear DNA area upstream of the gene (HiRd) P5 for acid sequence (1000 bp) TCACTTAATTCAAGCGCATCAATGTTTTCTAAAACATCAACAGAATTGACCGCACTTGTA TCTAAAATTTCGCCATTTATTAAGACTGCGCGTAATGCCAAAACATGATTAGAGGTTTTA CCATATTGCAATGAGCCTTGCCCAGAGGCATCGGTGTTAATCATTCCACCTAAAGTCGCT CGATTGCTGGTGGACAGTTCTGGGGCAAAGAACAAACCATGTGGTTTTAAAAATTGATTA AGTTGATCTTTTACTACGCCTGCTTGTACTCGAACCCAACGTTCTTTTACATTGAGTTCT AAGATGGCTGTCATATGACGAGAAAGATCCACTATTATATTGTTATTGATGGATTGCCCA TTTGTGCCAGTGCCTCCACCGCGAGGCGTAAAGCTGATTGATTGATATTCAGGTAAATTT GCCAATTTTGTTATCCGCACTATATCAGCAACCGTTTTCGGAAAAAGAATTGCTTGTGGA AGTTGTTGGTAAACGCTGTTATCCGTAGCCAGACTTAATCTATCTGCATAGTTTGTCGCA ATATCCCCCTCAAAATGTTGGCATTGAAGATCATCAAGATAATCAAGTACATATTGTTCA ACTTGAGGAATGCGATTTAGATTTGGCAAC ATAGTATTTGACCCATTTAAACATATCAGA TGGAGGCTTTGATAATATCCTAAGGCTAGAATAATGTCGATTAGGAAAGAGAGAGGAGAA AGTAAAAAGTCTGTTTAAGAAAGTGTTATTTTGGATAAAAACTAAACAAAAAATTCAAAA GAATTTGATCTTTTCAATTTTTATAGGATAATAAGCGCACTTTTGAACGTTCCTTTGGGG TAAACATAAGCAAAGGAATTGAATTTGTCAAAAGGTAATAAAGTAGGGCAAATTCAAAAC CCTAGTTAAGTGACTGTTTATAATGTAGCTTTAATTAAAAGTTCAGTATAAACAAGGACA CTTTTTATTACTATTCGATCACTAAATAGAGGACATCAAAA -111 - This paper scale applicable Chinese National Standard (CNS) A4 size (210 x 297 mm) 1297731 A7 B7 V. description of the invention (109) Ministry of Economic Affairs Intellectual Property Office employees consumer cooperatives printed SEQ . ID NO: 68 H. influenzae (HiRd), nuclear Chang acid sequence (1000 bp) DNA region upstream of the D15 gene TCGATTGTATCCTATATAAATTATAGACGTAAAAAATCATTAAATAATGCAAACACCGTT AAGCTTAATAACAGTGCTGCGCCAATTCGATAACAGATGCTTTGCACCCGCTCAGAAACA GGTTTTCCTTTAACAGCTTCCATTGTTAAAAAAACTAAATGACCGCCATCTAATACTGGT AATGGAAATAAATTCATAATCCCTAAATTTACACTAATCAATGCCATAAAACTTAAAAAA TACACCAATCCAATATTTGCTGATGCGCCAGCACCTTTTGeAATAGAAATTGGCCCACTT AAATTATTTAATGACAAATCGCCAGTAAGTAATTTCCCTAATAT . TTTCAAGGTTAAAAGG GAAAGCTGTCCTGTTTTTTCAATGCCTTTTTGTAAAGATTCAAGAATACCATATTTTAAT TCAGTACGGTATTCATCCGCTAATTTTGTTAAGGCTGGGCTAACCCCAACAAACCATTTG CCATTTTGATTACGCACTGGAGTTAGGACTTTGTCAAATGTTTCTCCATTACGTTCAACT TTAATAGAAAAAGATTCGCCTTGTTCGACCTGTTTTATAAAATCTTGCCAAGGAAGTGCG GTTAAATTTTCTTTTAAAATTTTATCACCGATTTGTAAACCAGCTTTCTCAGCGGGAGAA TTTTGAACAACTTTAGAAAGCACCATTTCAATTTTAGGACGCATAGGCATAATCCCTAAT GCCTCAAAAGCACTTTCTTTTTCAGGATCGAATGTCCAATTTGTAAGATTTAAAGTCCGT TGTTGTTCAATATTAGAATTGAAAGGAGAAAGGCTAATCTCAACATTAGGCTCCCCCATT TTTGTGGCAAGTAGCATATTGATGGTTTCCCAATCTTGAGTTTCTTCGCCATCAATTGTA AGAATTTGCGTATTGGGTTCAATGTGGGCTTGTGCTGCGATTGAGTTTGGTGTTATTGAT TCAATCACTGGTTTAACCGTTGGCATTCCATAAAGGTAAAT SEQ ID NO: 69 H. influenzae (HiRd), nuclear Chang acid sequence of Omp26 gene upstream of the DNA region (1000 bp) TTTGATAAATATCCTTAATTAAATGATGGGTTTAATATTTTCTCTGCCCAATTAAATTAG GCAGAGAACGTTGTTTTTGAGTTCTGATGAAGAAAAAAGTTCAATTTATTAGAAAGAACC TCCAATACTAAATTGGAACTGTTCGACATCATCATTTTCATATTTTTTAATTGGTTTGGC ATAAGAGAATACCAATGGCCCAATAGGAGATTGCCATTGGAATCCGAC ACCTGTAGAGGC GCGAATACGGCTTGATTTGCCATAATCGGGTAAGCTTTTTAATACATTGTTATCTAACCC ACTCTTATCCGATTTCCACTTAGTATTCCAAACACTTGCCGCATCAACAAATAGGGAGGT TCGGACTGTATTTTGGCTTTTATCACTCACAAACGGTGTTGGTACAATAAGTTCTGCACT CGCAGTTGTGATTGCATTACCACCAATCACATCAGAACTTATCTTCTTAAAAGTACCATT ACCATTACCATGTTCTGCATAAATTGCGTTAGGTCCAATACTACCATAAGCAAAACCACG TAATGAACCGATGCCACCCGCTGTATAAGTTTGATAGAACGGTAAACGCTTGTTTCCAAA ACCATTTGCATATCCTGCAGATGCTTTT ^ CAGATACAACCCAGAGGTGATCTCTGTCTAA TGGGTAGAAACCCTGTACGTCTGCACTTAGTTTGTAGTATTTGTTATCAGAACCTGGAAT AGTAACTCGTCCACCAAGACTTGCTTTAACCCCTTTAGTTGGGAAATAGCCTCTATTAAG GCTGTTATAGTTCCAACCAAAAGAAAAATCAAAGTCATTTGTTTTAATGCCATTACCTTT AAATTTCATTGATTGAATATATAAATTACGGTTATATTCTAGAGCAAAGTTACTAATTTT ATTATAGGTATGGCCTAATCCTACATAATAGGAGTTATTTTCATTTACAGGGAAACCTAA AGTAACATTACTTCCATAAGTCGTACGCTTATAGTTAGAGG SEQ ID NO:. 70 i H. influenzae oath acid sequence (HiRd), DNA regions upstream of the P6 gene bad iKiOOO bp) TTAGATTTCTCCTAAATGAGTTTTTTATTTAGTTAAGTATGGAGACCAAGCTGGAAATTT AACTTGACCATCACTTCCTGGAAGGCTCGCCTTAAAGCGACCATCTGCGGAAACCAATT . G TAGCACCTTTCCTAAGCCCTGTGTAGAACTATAAATAATCATAATTCCATTTGGAGAGAG GGTTGGGCTTTCGCCTAGAAAAGATGTACTAAGTACCTCTGAAACGCCCGTTGTGAGATC TTGTTTAACTACATTATTGTTACCATTAATCATCACAAGTGTTTTTCCATCTGCACTAAT TTGTGCGCTACCGCGACCACCCACTGCTGTTGCACTACCACCGCTTGCATCCATTCGATA AACTTGTGGCGAACCACTTCTATCGGATGTAAATAAAATTGAATTTCCGTCTGGCGACCA CGCTGGTTCAGTATTATTACCCGCACCACTCGTCAATTGAGTAGGTGTACCGCCATTTGC TCCCATAACGTAAATATTCAGAACACCATCACGAGAAGAAGCAAAAGCTAAACGAGAACC ATCTGGCGAAAAGGCTGGTGCGCCATTATGCCCTTGAAAAGATGCCACTACTTTACGTGC GCCAGAATTTAAATCCTGTACAACAAGTTGTGATTTTTTATTTTCAAACGATACATAAGC CAAACGCTGGCCGTCTGGAGACCAAGCTGGAGACATAATTGGTTGGGCACTACGATTGAC GATAAATTGATTATAGCCATCATAATCTGCTACACGAACTTCATAAGGTTGCGAACCGCC ATTTTTTTGCACAACATAAGCGATACGAGTTCTAAAGGCACCACGGATCGCAGTTAATTT TTCAAAAACTTCATCGCTCACAGTATGCGCGCCATAGCGTAACCATTTATTTGTTACTGT ATAGCTATTTTGCATTAATACAGTCCCTGGCGTACCTGATGCACCAACCGTATCAATTAA TTGATAAGTAATACTATAACCATTACCCGATGGAACCACTT SEQ ID NO: 71 (H. influenzae (non-type), the nuclear DNA area TbjiA travel gene Mi Chang acid sequence 1000 bp)

CAAAATTA CAAAAACC

TCCAGAGAAACCTTAATTGATGGCAAGCTAACTACTTTTAAAAGAACTGATGCAAAAACC AATACAACAGCCGATACAACAACCAATAAAACAACCAATGCAATAACCGATGAAAAAAAC TTTAAGACGGAAGATATACTAAGTTTTGGTGAAGCTGATTATCTTTTAATTGACAATCAG CCTGTTCCGCTTTTACCTGAAAAAAATACTGATGATTTCATAAGTAGTAGGCATCATACT GTAGGAAATAAACGCTATAAAGTGGAAGCATGTTGCAAGAATCTAAGCTATGTAAAATTT -112- This paper scales applicable Chinese National Standard (CNS) A4 size (210 X 297 mm) on the back of the green stains r tonic? Please fill in this page again) ▼Install-----r---tT--------- 1297731 A7 B7 V. Description of invention (

, GAAGAAAAA &lt; CAAGAAAAAAAAGAAAAAGAAAAACAAACGACGACAACATCTATCGAGACTTATTATCAA TTCTTATTAGGTCACCGTACTGCCAAGGCCGACATACCTGCAACGGGAAACGTGAAATAT CGCGGTAATTGGTTTGGTTATATTGGTGATGACACGACATCTTACTCCACTACTGGAGAT AAAAATGCTCTCGCCGAGTTTGATGTAAATTTTGCCGATAAAAAGCTAACAGGCGAATTA AAACGACACGATAATGGAAATACCGTATTTAAAATTACTGCAGACCTTCAAAGTGGTAAG AATGACTTCACTGGTACAGCAACCGCAACAAATTTTGTAATAGATGGTAACAATAGTCAA ACTGGAAATACCCAAATTAATATTAAAACTGAAGTAAATGGGGCATTTTATGGACCTAAG GCTACAGAATTAGGCGGTTATTTCACCTATAACGGAAATTCTACAGCTAAAAATTCCTCA ACCGTACCTTCACCACCCAATTCACCAAATGCAAGAGCTGCAGTTGTGTTTGGAGCTAAA TAATGG GGTATGTATTATGAAGACCCACTTAAAGAAi

GAAAAAGAAAAAGAAAAAGAC AAACAACAAGTAGAAACAACCAAGT^

SGAATACTAAAAA SEQ ID NO:. 72 H. influenzae (HiRd), nuclear Chang acid sequence (1000 bp) DNA region upstream of the gene TbpB, TAGAATTATATTCTTATACAAAATTGATAATTGTTCGCATTATCATTTTTTTTTTGTAAT AATGTCAACTTATAATTTTTTAAGTTCATGGATAAAATATGAAAAATGGCGTAAAACAAC TTTTTCTCTTATCATTAATAGGCTTATCATTAACGAATGTAGCTTGGGCAGAAGTTGCAC CAAAGTGCGGAATTAAAAACCTCCTCTT ^ AAAAGC TCAAAAT CAACTGTTATTACCACTATATAAACAATTTCCTCAACAAGATAATT TCTTACTAACTTGGGCAAAGGC TTGCTTATTATCGTGAATTATTCGCTCGAGACGCATCTTTACTACCTTTACGTTATTAAT TAGCTCAAGCTCTATTTTTTAACTATGAAAATGAAGCTGCCAAAATTCAATTTGAAAAAT TACGTACAGAGGTAGATGATGAAAAATTTTTAGGTGTTATTGATCAGTATCTTTTAACAC TAAATCAGCGGAATCAATGGATATGGCAAGTAGGATTAAATTTTTTAAATGATGATAATT TGAATAACGCTCCAAAAAGTGGCACAAAAATTGGTAGTTGGACCGCTTGGGAAAAAGAAA GTGGGCAGGGGGTAGGGTATTCTTTATCAGTAGAAAAAAAATGGCCATGGGCAGATCATT TTTTTAGTAAAACTATGTTTAATGGGAATGGAAAATATTATTGGGATAATAAAAAATACA ATGAGGCTACTGTGCGTATAGGTGGTGGTTTAGGCTATCAAACTGCCTCAGTTGAAGTCT CGTTGTTTCCTTTTCAAGAAAAACGCTGGTATGCAGGCGGT SEQ ID NO: 73 Haemophilus influenzae (LKP serotype 1 genome) , HifA (cilia hormone) area for nuclear DNA upstream acid sequence (1000 bp) GTCCTAAAAATGATACATT (GACAAA · AGAAATi .TACGATTCfl

TTTCCTCTATGCCTAAGAAAGAAATACCAAATAGGCATATTATTTCTCTTTCCAGAGCC AATTAGCGCACCATCCAAGGCTTGTTTTGCGTGGGTTAATTCCTGCTTTATATCAAAATA ACACTCAGGCAGTTCAACTGTTATTACCACTATATAAACA 3GCTATTGAAGCTCGTGAACAAGGTGATTTAACTCAATCTA I----- (Please read the note on the back and fill out this page)

AGAGGTT CAAAGGC order ---

AACCGTTTATTAAAATGCCAAAGGCTTAATAAACAGCAAACTTTGTTTTCCCAAAAAAAG TAAAAAACTCTTCCATTATATATATATATATATATAATTAAAGCCCTTTTTGAAAAATTT CATATTTTTTTGAATTAATTCGCTGTAGGTTGGGTTTTTGCCCACATGGAGACATATAAA AAAGATTTGTAGGGTGGGCGTAAGCCCACGCGGAACATCATCAAACAACTGTAATGTTGT ATTAGGCACGGTGGGCTTATGCCTCGCCTACGGGGAAATGAATAAGGATAAATATGGGCT TAGCCCAGTTTATGGATTTAATTATGTTGAAATGGGGAAAACAATGTTTAAAAAAACACT TTTATTTTTTACCGCACTATTTTTTGCCGCACTTTGTGCATTTTCAGCCAATGCAGATGT GATTATCACTGGCACCAGAGTGATTTATCCCGCTGGGCAAAAAAATGTTATCGTGAAGTT AGAAAACAATGATGATTCGGCAGCATTGGTGCAAGCCTGGATTGATAATGGCAATCCAAA TGCCGATCCAAAATACACCAAAACCCCTTTTGTGATTACCCCGCCTGTTGCTCGAGTGGA AGCGAAATCAGGGCAAAGTTTGCGGATTACGTTCACAGGCAGCGAGCCTTTACCTGATGA TCGCGAAAGCCTCTTTTATTTTAATTTGTTAGATATTCCGCCGAAACCTGATGCGGCATT TCTGGCAAAACACGGCAGCTTTATGCAAATTGCCATTCGCTCACGTTTGAAGTTGTTTTA TCGCCCTGCGAAACTCTCGATGGATTCTCGTGATGCAATGAAAAAAGTAGTGTTTAAAGC CACACCTGAAGGGGTGTTGGTGGATAATCAAACCCCTTATTATATGAACTACATTGGTTT GTTACATCAAAATAAACCTGCGAAAAATGTCAAAATGGTTG Φ. Ministry of Economic Affairs Intellectual Property Office consumer co-workers Printing

SE〇 ID NO:. 74 H. influenzae (the LKP serotype 1 genome), HifE (cilia tip element) Nuclear Chang acid sequence (looo bp) DNA region upstream of TAGTAGATTTCCGCACGGGCAAAAATACAATGGTGTTATTTAACCTCACTTTGCCAAATG GCGAGCCAGTGCCAATGGCATCCACCGCACAAGATAGCGAAGGGGCATTTGTGGGCGATG TGGTGCAAGGTGGTGTGCTTTTCGCTAATAAACTTACCCAGCCAAAAGGCGAGTTAATCG TCAAATGGGGTGAGCGAGAAAGCGAACAATGCCGTTTCCAATATCAAGTTGATTTGGATA ACGCACAAATACAAAGTCACGATATTCAATGCAAAACCGCAAAATAAATAATTGAAGAGG ATTTATGCAAAAAACACCCAAAAAATTAACCGCGCTTTTCCATCAAAAATCCACTGCTAC TTGTAGTGGAGCAAATTATAGTGGAGCAAATTATAGTGGCTCAAAATGCTTTAGGTTTCA TCGTCTGGCTCTGCTTGCTTGCGTGGCTCTGCTTGATTGCATTGTGGCACTGCCTGCTTA TGCTTACGATGGCAGAGTGACCTTTCAAGGGGAGATTTTAAGTGATGGCACTTGTAAAAT TGAAACAGACAGCCAAAATCGCACGGTTACCCTGCCAACAGTGGGAAAAGCTAATTTAAG CCACGGAGGGCAAACCGCCGCCCCTGTGCCTTTTTCCATCACGTTAAAAGAATGCAATGC AGATGATGCTATGAAAGCTAATCTGCTATTTAAAGGGGGAGACAACACAACAGGGCAATC TTATCTTTCCAATAAGGCAGGCAACGGCAAAGCCACCAACGTGGGCATTCAAATTGTCAA AGCCGATGGCATAGGCACGCCTATCAAGGTGGACGGCACCGAAGCCAACAGCGAAAAAGC CCCCGACACAGG TAAAGCGCAAAACGGCACAGTTATTCAACCCCGTTTTGGCTACTTTGG CTCGTTATTACGCCACAGGTGAAGCCACCGCAGGCGACGTTGAAGCCACTGCAACTTTTG AAGTGCAGTATAACTAAAATATTTATTATCCAGTGAAAAAA -113- This paper scale applies Chinese National Standard (CNS) A4 specification (210 297 297 public) 1297731 A7 B7 V. Invention description (111) SEQ. ID NO: 75 Haemophilus influenzae (HiRd), Acid sequence of the upstream DNA region of the P2 gene ii〇〇〇bp) 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 TTATCCGCTA ATCANCGGGG AACGACGATT TCATAATGCG AATTTGGCGA AATACTTGCC AAATTTTTAC GGTGCCGTAT TTTCCAAACC TAAATACATT TATATGAAAA TTGGAAAGCA CCTTAGATGA GGCCATGCTG AGGGGCAGAA ACAACTGATA AAACCTAAAA TCGATCAGAT TTTTTTTGTA TGGAGAAAAG A ;

ACATTTCATC CGATCTGGCT TATTAATTCA CGCCAAGCTG TTGGGATTAA ATTCTACACA TGATGGATCT TGCGTTATAA ACCAATAATC AAAAGAACCT ATGGCATAAC AGTTCTGGAA ATCCATCCAA GACACAGAGA AATCCGACAT TAACGTCATA CAGAATAAAA TTCAAGAAAA AAGATGCAGC TTCAATCATA

AGTAATTCCA TAATATAAAT ACTGCACCAA ATTCATACCT TCATTTGTTC ACAACCTTTT TGCTTAGGTA ACAACATGAA GAATGGAATT TGCTTGATCA CAAATGGATC AGCCTGTAAA CGTCATJ AAATG; TGGAAGATAT TTTTTGGATA AATAATCAAA TTAAAATTTT TCGTCCGTTT GATAGTAAAC

ATAAAA GAAATT

TGAACTTTAA ATGAYAATTA TTTCAATAAT GTAGTTTCAG AACCTATCTC TGTTATGCCK ATCCAGGGGC AAAACACTCT ACGCGCTGTC CGCTTTATAG TTTAACTGGA AAACCAAGAT TTAATTGGCA TGCGATGAAT GGGGTACATA CCTAAAAATA TTCATTTAAA GGAGTATTGA TGGCGATTGG AACCATAAGG

TCGCATCAGG TTACCTGTGT GCAGTGTCCT TATCTAATAC TTTCCATTAA AAACAGATTG GGGCGGAATT IGCTA GCAT TGATAGCCAA AAAAAAATAA TTATGGATAG ATGGGTAAAA TAGCAAAAAA GAAGAATAAT TTTAATACTT AAATGTGATC CATCAAAAAT ACAATTCTAT AATACAAATT

AAA CC CC CC CC CC CC

TCAGTGCTTG GGCGGCGTGT GCAGCATCCA CAGCGGCAAA AGTCGACAGT AAATT; CCAAG1 GAATGCTTGG TCAAAAATGC AATGCCAGCC AACACTCAAA ATCCATCAGG AGTACGCTGG CTTAAGCTGT ATGAATTAAA ACTGCTTTAA TTATATGTGG CTTATTTACT GCCATGTCAA

ΓΑΑΑΑΟ 3TGGTA

GTTTTTTAAG GCGTCTTATA AGCCAATTTA AACTCGCCAA CTTAAAACTT GGTTCATCAT TGCTTGCCAT CTCAATACCT GGCGGTAGAT TTGTACCCAC GCAGGTGGAT TGGTGAGATT CGTCAAAGCT ATTCGGCGTG TGATGAACAA ATGGTGCGAT AGCTATCGTC TAATGAAAAT AGGATAGTTA

ATATGTACCG TTTCCTATCA ATCTTGGTTC ACAAATCCTA GGGCAATGCC GAAGATATCC TGTGCCTCAT TTGGCTCCCC CGCTTTGTTT AGATGCTAGT TTAGTATCAT GCTCCTTTTT TGCTGCAAAA AAGATGGCGA CTTTATTCAA GGAACAAATG GGTTCAAGCA GAGCTAAAAA TGAG

CTGTCAGTCC TTGCAGGCTT AAGAT .TCAGC ACCAAAATGG TAATCAAAGC ATCGGCACTT TACTATCATG TACAGGGGCG GGTGTTAAGG ACTGCCCGAC TTGGTATTAA ACTGGTTGTG TGGTTTTGAA AAAATACCAA ATTTATCCAC TACACCACTA AAATAGCCAT

ACCCCA AAAAAT

TGCATGGATT AGTATTTATC GCCAGACGCA TCATCAGTGC TCTATCGCAC ACTTGCCAAT GGGAGATGAT TATAAGCCCA TGAAAGACTA CAATTTTTAA CATGTACCTG AACCCTAACC CTCTTGTTGG ATTTTTTGTT AATTGCAACC ATTTTTTGCA lTAATC ICTTCAA

TTAAAT AAAGCT protein sequence: 25% homogeneity and 35% similarity with E.coli winter HtrB l·----------装—^—订--------- (please first Read the notes on the back and fill out this page. Printed by the Intellectual Property Office of the Ministry of Economic Affairs

1 SVLGFLRYVP 51 QRQKLAKQIL 101 PSGMLAIVPH 151 NASLVPTDAS 201 STLASKLAAK 251 TALNGAMEQM 301 AMSKDSYE

LSVLHGLAAC KNQLISAVDS IGTWEMMNAW GVKAIFKTLK TGCALVGLSC IYPHFLHYMW

ASYISYHCRL LKTWAMPPKW LNTFGSPTIM AGGFSIILPD IRREDGDGFE SYRRFKHTPL

SIYRSIQANL SIAQIKTVHH YKPIKNAAVD HVPDPSGGEI IFCYELNDEQ LNNPYLLNEN

ILVHPKMPDA EDILIKALAN RFVLQGRERL APFFGIKTLT LYSKNTKIAT ELKKIAIKLQ SEQ. ID NO: 77 Neisseria (meningococcal B) The nucleotide sequence of the HtrB gene DNA coding region ' 1 ATGTTTCGTT 51 CATCCTGTTG 101 GTCTGCACAt 151 AAGGAAGACC 201 TCCCGACCCC 251 GTTTGG; 301 ATGTTC; 351 ACACGAAGGG 401 GCGGACGCTA 451 AAACCGCCGA 501 TCGC 551 TCATC.

GAACT CAAAG

GGCAAA CAAAGC

TACAATTCGG ACCGCCCTGC GCTGGGAAAC GCGCGCGCAT AAAACAGTCA TGCCCCCGCG CGGTACACGG CTGCTATTCA CATCAGCCAG AAATCAAAGC GGAAAAACCG CCTGCGTTCG

GCTGTTTCCC TCAAATGCCT CGGCTCGGAC CGTCGCCAAT AAGCCGTTTT TTTTTCAGAA CTGGGAACAT TCACGCCGCA CAGCTTCCGT GATAGACAAA CGCCTACCAG GGCGAAGCAA CCTTTGCGAA CTCCCTGCTG ATCTGGCGTT ATGCGTCAGG TGCGGAAACG AACCGGAAGA GTGCAGCAGG CATCGGCAGC TCCCGCTGAC ATCATGCAGG CATACAAGGG CCATCGTCCT-114-

CCGCCATGCA CCACTTTCCT TTACCTTTTA CAGGCATGAA GCAAAAGGCG CATAGAAACA CTTTGGACAA TACGATTTGG CGCCATGTAC CGGGCAGGGT GTCAAACAAA GCCCGACCAC This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1297731 A7 B7 V. Description of invention (112)

601 GTCCCCTCCC 651 CAAACCTGCC 701 GCGTGAAAAC 751 TTCGATTTGC 801 CCATGATGCC 851 TTCCGACGCA

CTCAAGAAGG TATACCATGA CCTGTTTTTC ACATCCGCCC GCCGTGTTCA GTATCTGTTT

CGGGGAAGGC CGCTGGCGGC TGCTGCGAAC CGTCCAAGGG ACCGCAATGC ATGTACAACC

GTATGGGTGG AAAATTGGCA GCCTGCCTGG GAATTGAACG CGAATATTGG GCTACAAAAT

ATTTCTTCGG CACGTCAAAG CGGACAAGGT GCGACAAAGC ATACGCCGTT GCCG protein sequence is 30% homogenous and 38% similar to Htrb Ε· coli

1 MFRLQFGLFP 51 KEDRARIVAN 101 MFKAVHGWEH 151 KPPKIKAIDK 201 VPSPQEGGEG 251 FDLHIRPVQG

PLRTAMHILL· MRQAGMNPDP VQQALDKHEG IMQAGRVRGK VWVDFFGKPA ELNGDKAHDA

TALLKCLSLL KTVKAVFAET LLFITPHIGS GKTAPTSIQG YTMTLAAKLA AVFNRNAEYW

PLSCLHTLGN AKGGLELAPA YDLGGRYISQ VKQIIKALRS HVKGVKTLFF IRRFPTQYLF

RLGHLAFYLL FFRKPEDIET QLPFPLTAMY GEATIVLPDH CCERLPGGQG MYNRYKMP

SEQ. ID NO: 78 H. pylori (undifferentiated) HtrB gene DNA coding region Nucleic acid sequence AAAAAC TTTTGG 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901

ATGAAAAACG AAAAACTCCC TCAATTTCAA CCGCACTTTT TAGCCCCAAA ATACTGGCTT TTTTGGCTAG GCGTGGCAAT TTGGCGAAGT ATTTTATGTC TTCCCTATCC TATTTTGCGC CATATTGGTC ATGGTTTCGG TTGGCTGTTT TCACATTTAA AAGTGGGTAA ACGTCGAGCT GCCATTGCAC GCCGTAATCT TGAACTTTGT TTCCCTGATA TGCCTGAAAA CGAACGTGAG ACGATTTTGC AAGAAAATCT TCGTTCAGTA GGCATGGCAA TTATCGAAAC TGGCATGGCT TGGTTTTGGT CGGATTCACG TATCAAAAAA TGGTCGAAAG TTGAAGGCTT ACATTATCTA AAAGAAAATC AAAAAGATGG AATTGTTCTC GTCGGTGTTC ATTTCTTAAC GCTAGAACTT GGCGCACGCA TCATTGGTTT ACATCATCCT GGCATTGGTG TTTATCGTCC AAATGATAAT CCTTTGCTTG ATTGGCTACA AACACAAGGC CGTTTACGCT CCAATAAAGA TATGCTTGAT CGTAAAGATT TACGCGGAAT GATCAAAGCT TTACGCCACG AAGAAACCAT TTGGTATGCG CCTGATCACG ATTACGGCAG AAAAAATGCC GTTTTTGTTC CTTTTTTTGC AGTACCTGAC ACTTGCACTA CTACTGGTAG TTATTATTTA TTGAAATCCT CGCAAAACAG CAAAGTGATT CCATTTGCGC CATTACGCAA TAAAGATGGT TCAGGCTATA CCGTGAGTAT TTCAGCGCCT GTTGATTTTA CGGATTTACA AGATGAAACG GCGATTGCTG CGCGAATGAA TCAAATCGTA GAAAAGGAAA TCATGAAGGG CATATCACAA TATATGTGGC TACATCGCCG TTTTAAAACA CGTCCAGATG AAAATACGCC TAGTTTATAC GATTAA protein sequence is 57% homogenous and 66% similar to HtrB E. coli

1 MKNEKLPQFQ 51 SHLKVGKRRA 101 WFWSDSRIKK 151 GIGVYRPNDN 201 PDHDYGRKNA 251 SGYTVSISAP 301 RPDENTPSLY PHFLAPKYWL AIARRNLELC WSKVEGLHYL PLLDWLQTQG VFVPFFAVPD VDFTDLQDET D*

FWLGVAIWRS FPDMPENERE KENQKDGIVL RLRSNKDMLD TCTTTGSYYL AIAARMNQIV

ILCLPYPILR TILQENLRSV VGVHFLTLEL RKDLRGMIKA LKSSQNSKVI EKEIMKGISQ

HIGHGFGWLF GMAIIETGMA GARIIGLHHP LRHEETIWYA PFAPLRNKDG YMWLHRRFKT SEQ. ID NO: 79 The nucleotide sequence of the DNA coding region of the MsbB gene of Haemophilus influenzae (undifferentiated) 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951

ATGTCGGATA AGCGCACTTT TTGGTATTTT GATAAATTGA ACAGCGTACG CTGAACAACA CAGGTTATGT GCAAAAACGC CTGAAGGAAA GCGTCTGGCA TAATCCACAC AACGTTTCGG TTAAGTCATG TTTTGGGGCG CGACATTACC ATTCCAATGT AATTCATCCT CAATGAACGA GTTTGGATTT TGATTAA

ATCAACAAAA TCATGGTCGT CTTTTTATTG CGGGAAAATT CGTGCACAAA ACGTGAGCAA TTGGTATTGG AGCGAATTTA GAATATTATT TTATTTTGCA CGTAATCCAT CGGAAAAATG TTCGTAAAGG GAACAAAGCG AGGGTTAAAT TTCCTCGTTA GCAATGAATT AGAAATAGAA TGCAATTATT

TTTACGTTTG ATTTAAAGCC TTGTTAGCAT AGGTATTTGG CTAACTTGCA GTGATTGATA TGAGATTGCC TCGGTCTTGA CTTATGGTGC CACTCAAGGC TGGTGGATTG CATGCACGCC CGAAATGGGT TATTTGTTGA .TGGCAA .CGCTGAA TAAGTGATGA TCTTTTGTTA GCGTACAAGG (Please read the back note and fill out this page) 严-----r---IT----- ----^^1.

ACGGCGAGAG TCAATATTGG TTGTGCCTTT ATTGGGCATA ATATTGTTTC AAATGTTTGC ATCCGTTCAA ACATATCGAA CACATGGCTG ATGCCAATGA GCTTTGGACG AAAATGGTAT TATTACTTAC TTTCTTTGGG AACTTTCTAA ACGGGCAAAT TCCTGAACAA CGCCAGCGCC AAAGATGGCG

TGGGCTATGA GGGATTTGGC TCGTCTGCGC AAGCAAAGAA CCTCATTGGA GGTTGTCGCT AGAAACATTT CAGGCAAAAG GGCGATTGAT CTTCTATGTA ATTACACGCC TAAACCTTTT CCGATGAAGA ACTTATAAAG AGCCGTTGTT ATGAAATGGA TCAGCCCGAG AGAGCAATAT AAGATCTTTA Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed -1 _

AAAAT TAACG Protein Sequence - 45% with MsbB E. coli ^] qualitative similarity and 56% 1 MSDNQQNLRL TARVGYEAHF SWSYLKPQYW GIWLGIFFLL LLAFVPFRLR 51 DKLTGKLGIW IGHKAKKQRT RAQTNLQYCF PHWTEQQREQ VIDiCMFAVVA 101 QVMFGIGEIA IRSKKHLQKR SEFIGLEHIE QAKAEGKNII LMVPHGWAID 151 ASGIILHTQG MPMTSMYNPH RNPLVDWLWT ITRQRFGGKM HARQNGIKPF -115-! This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1297731 A7 B7 V. Invention description (113) 201 LSHVRKGEMG YYLPDEDFGA EQSVFVDFFG TYKATLPGLN KMAKLSKAVV 251 IPMFPRYNAE TGKYEMEIHP AMNLSDDPEQ SARAMNEEIE SFVTPAPEQY 301 VWILQLLRTR KDGEDLYD* Ministry of Economic Affairs Intellectual Property Bureau Printed by employee consumption cooperatives

SEQ. ID NO: 80 Nucleic acid sequence of the DNA coding region of M. catarrhalis MsbB gene 1 ATGAGTTGCC ATCATCAGCA TAAGCAGACA CCCAAACACG CCATATCCAT 51 TAAGCATATG CCAAGCTTGA CAGATACTCA TAAACAAAGT AGCCAAGCTG 101 AGCCAAAATC GTTTGAATGG GCGTTTTTAC ATCCCAAATA TTGGGGAGTT 151 TGGCTGGCTT TTGCGTTGAT TTTACCGCTG ATTTTTCTAC CGCTGCGTTG 201 GCAGTTTTGG ATCGGCAAGC GTCTTGGCAT TTTGGTACAT TACTTAGCTA 251 AAAGCCGAGT TCAAGACACT CTAACCAACC TGCAGCTTAC CTTCCCAAAT 301 CAACCAAAAT CAAAACACAA GGCCACCGCA CGGCAAGTAT TTATTAATCA 351 AGGTATTGGT ATTTTTGAAA GTTTATGTGC ATGGTTTCGC CCTAATGTCT 401 TTAAACGCAC TTTTAGCATT TCTGGTTTAC AGCATTTGAT TGATGCCCAA 451 AAACAAAATA AAGCGGTGAT TTTACTTGGT GGACATCGCA CGACGCTTGA 501 TTTGGGCGGT CGGTTATGTA CACAGTTTTT TGCGGCGGAC TGCGTGTATC 551 GCCCACAAAA CAACCCTTTG CTTGAATGGT TTATCTATAA TGCACGCCGC 601 TGTATCTTTG ATGAGCAAAT CTCAAATCGT GATATGAAAA AACTCATCAC 651 TCGGCTCAAA CAAGGTCGGA TAATTTGGTA TTCACCTGAT CAAGATTTTG 701 GTCTTGAGCA TGGCGTGATG GCGACCTTTT TTGGTGTGCC TGCAGCAACG 751 ATTACCGCTC AGCGTCGTCT TATTAAGCTG GGTG ATAAAG CCAATCCTCC 801 TGTCATCATC ATGATGGATA TGCTCAGACA AACGCCCGAT TATATCGCAA 851 AAGGTCACCG TCCACATTAT CACATCAGCC TAAGCGCTGT GTTAAAAAAT 901 TATCCCAGCG ATGACGAAAC CGCCGATGCT GAACGCATCA ATCGACTGAT 951 TGAGCAAAAT ATTCAAAAAG ATTTAACCCA GTGGATGTGG TTTCATCGCC 1001 GCTTTAAAAC TCAAGCCGAT GACACCAATT ACTATCAACA TTAATG protein sequencer bad 'l · E. coli MsbB with 28 and 37% homogeneity % similarity 1 MSCHHQHKQT PKHAISIKHM PSLTDTHKQS SQAEPKSFEW AFLHPKYWGV 51 WLAFALILPL IFLPLRWQFW IGKRLGILVH YLAKSRVQDT LTNLQLTFPN 101 QPKSKHKATA RQVFINQGIG IFESLCAWFR PNVFKRTFSI SGLQHLIDAQ 151 KQNKAVILLG GHRTTLDLGG RLCTQFFAAD CVYRPQNNPL LEWFIYNARR 201 CIFDEQISNR DMKKLITRLK QGRIIWYSPD QDFGLEHGVM ATFFGVPAAT 251 ITAQRRLIKL GDKANPPVXI MMDMLRQTPD YIAKGHRPHY HISLSAVLKN 301 YPSDDETADA ERINRLIEQN IQKDLTQWMW FHRRFKTQAD DTNYYQH * SEQ. ID NO :81 Neisseria (meningococcal 0) M sb B gene DNA coding region of nucleotide sequence 1 ATGAAATTTA TATTTTTTGT ACTGTATGTT TTGCAGTTTC TGCCGTTTGC 51 GCTGCTGCAC AAACTTGCCG ACCTGACGGG TTTGCTCGCC TACCTTTTGG 10 1 TCAAACCCCG CCGCCGTATC GGCGAAATCA ATTTGGCAAA ATGCTTTCCC 151 GAGTGGGACG GAAAAAAGCG CGAAACCGTA TTGAAGCAGC ATTTCAAACA 201 TATGGCGAAA CTGATGCTTG AATACGGCTT ATATTGGTAC GCGCCTGCCG 251 GGCGTTTGAA ATCGCTGGTG CGTTACCGCA ATAAGCATTA TTTGGACGAC 301 GCGCTGGCGG CGGGGGAAAA AGTCATCATT CTGTACCCGC ACTTCACCGC 351 GTTCGAGATG GCGGTGTACG CGCTTAATCA GGATGTACCG CTGATCAGTA 401 TGTATTCCCA CCAAAAAAAC AAGATATTGG ACGCACAGAT TTTGAAAGGC 451 CGCAACCGCT ACGACAATGT CTTCCTTATC GGGCGCACCG AAGGCGTGCG 501 CGCCCTCGTC AAACAGTTCC GCAAAAGCAG CGCGCCGTTT CTGTATCTGC 551 CCGATCAGGA TTTCGGACGC AACGATTCGG TTTTTGTGGA TTTTTTCGGT 601 ATTCAGACGG CAACGATTAC CGGCTTGAGC CGCATTGCCG CGCTTGCAAA 651 TGCAAAAGTG ATACCCGCCA TCCCCGTCCG CGAGGCGGAC AATACGGTTA 701 CATTGCATTT CTACCCGGCT TGGGAATCCT TTCCGAGTGA AGATGCGCAG 751 GCCGACGCGC AGCGCATGAA CCGTTTTATC GAGGAACCGT GCGCGAACAT 801 CCCGAGCAGT ATTTTTGGCT GCACAAGCGT TTCAAAACCC GTCCGGAAGG 851 CAGCCCCGAT TTTTACTGAT ACGTAA protein sequences - and msbB E · coli has 25% homogeneity and 36% similarity 1 MKFIFFVLYV LQFLPFAL LH KLADLTGLLA YLLVKPRRRI GEINLAKCFP 51 EWDGKKRETV LKQHFKHMAK LMLEYGLYWY APAGRLKSLV RYRNKHYLDD 101 ALAAGEKVII LYPHFTAFEM AVYALNQDVP LISMYSHQKN KILDAQILKG 151 EINRYDNVFLI GRTEGVRALV KQFRKSSAPF LYLPDQD.FGR NDSVFVDFFG 201 IQTATITGLS RIAALANAKV IPAIPVREAD NTVTLHFYPA WESFPSEDAQ 251 ADAQRMNRFI EEPCANIPSS IFGCTSVSKP VRKAAPIFTD (Read precautions to fill out the back of the page) - means -----r---订--------- -116- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 public)

Claims (1)

YH^〇f9118838 Patent application, please use Naina to replace this (96 years Λο 1 B8 6 years &quot; month 4 repair (agricultural) original i November) S8 VI. Patent application scope 1 · A separation from the modification A genetically engineered large blister preparation of N. meningitidis strains, characterized in that the preparation is obtained by the following processes: a) reducing the variable or non-immunological advantage of the large blister preparation. A method of protecting an antigen comprising determining the nature of the antigen, genetically manipulating the bacterial strain to produce a less wild-type p 〇r A antigen or no Por A antigen, and making a large blister from the strain. 2. The genetically engineered large blister preparation according to claim 1 of the patent application, wherein the preparation is further obtained by one or more processes selected from the group consisting of: b) positively regulating large blister preparations with protection A method of expressing an omp antigen, the method comprising identifying the antigen, and genetically operating the strain to introduce a stronger promoter sequence upstream of the gene encoding the antigen such that the level of expression of the gene is higher than that of the unmodified large blister, and a large blister made in the strain; c) a method for positively regulating the expression of a protective OMP antigen in a large blister preparation, the method comprising identifying the antigen, and the genetically manipulated bacteria can be removed from the performance Control mechanism and make large blisters from this strain; d) Method for modifying the bacterial aps lipid a portion of the large blister preparation 'This method involves identifying the lipid a portion of the LPS to be toxic &lt; gene a step of genetically operating a bacterial strain to reduce or sever the performance of a child's gene and to make a large blister from the strain: e) modifying the bacterial lyse in the large blister preparation The method of the quality part a, this 65645-961123.doc small paper scale applies to the Chinese National Standard (CNS) A4 specifications (210X297 public) 1297731 έ88 C8 __·_ D8_ Six, the application for patent garden method including identification involves The lipid A moiety of LPS is less toxic than the genetically engineered strain to introduce a stronger promoter sequence upstream of the gene, such that the gene is expressed at a higher level than the unmodified large blister, and from the strain Medium water cancer; f) A method of reducing the toxicity of lipid A in large vesicular preparations and increasing the level of protective antigens. This method involves genetically manipulating the chromosomes of the strain to include the gene encoding the polymyxin A peptide. Or a derivative or analog thereof, fused to a protective antibody, and a large blister is prepared from the strain; g) a method of producing a retained 〇Mp antigen in a large blister preparation, the method comprising identifying This antigen, the strain is genetically manipulated to delete the variable region of the gene encoding the antigen, and a large water sore is made from the strain. h) a method for reducing the expression of antigens in large blister preparations, which shares structural similarities with human structures and which can induce autoimmune responses in humans, including identification of genes involved in antigen synthesis Genetically manipulating the strain to reduce or sever the performance of the gene and making large blisters from the strain; or 1) a method of positively regulating the expression of a protective QMP antigen in a large blister preparation, the method comprising identifying the antigen, Genetically engineered strains introduce more than one set of genes encoding the antigen in the chromosome and are controlled by a heterogeneous, strong promoter sequence and produce large water cancer from the strain. 3. According to the large blister preparations in the scope of patent application range i to 2, the party is 65645-961123.doc Λ L______-2- This paper scale applies to the Chinese National Standard (CNS) Α4 specification (210X297 mm) ABCD 1297731, application The genetic manipulation steps of patent range methods a), b), c), d), e), h) and i) are carried out by homogenous recombination, which occurs at least 3 nucleotides of the acid on the bacterial chromosome. 'and the transformation between the sequences of at least 3 nucleotides on the vector within the strain. 4. The large blister preparation according to item 3 of the patent application scope, wherein the transposition operation step is carried out by double cross-homogeneous recombination, and the event occurs on the bacterial chromosome by at least 3 separated by a nucleotide sequence, X | The two sequences of one nuclear fenic acid and two sequences of at least 3 nucleotides separated by the nucleotide sequence 'Y' on the vector transformed into the strain, wherein in the recombination X and γ is to be interchanged. 5. The large blister preparation according to item 4 of the patent application, wherein the two nucleotide sequences are about the same length, and wherein the carrier is a linear molecule. 6. The large blister preparation according to item 4 of the patent application, wherein the recombination of methods a) 'b), c), d), e) and h) is 1000 bp upstream of the start codon of the key gene Occurs within the area. 7. The large blister preparation according to item 6 of the patent application, wherein in the method a), d) or h) the acid sequence χ comprises a partial gene promoter and the (tetra) acid sequence Y comprises (iv) a promoter region Or no promoter region 8. According to the large (four) preparation of the fourth paragraph of the patent application, wherein the method and the recombination of h) are carried out, such that the nuclear (four) sequence includes the coding sequence of the desired gene. According to the patent application scope 4 of the large water cancer preparation, the method "65645-961123.doc" as much as the paper size _ 檩 ^ ^ Α 4_2ι () χ ^ ABCD 1297731 Sixth, the scope of the patent application "reorganization is carried out, Such a nucleotide sequence Y further includes the number of sets of genes that represent the sputum. 10. A large blister preparation according to claim 2, which can be prepared by applying methods b) and/or i), wherein one or more genes are positively regulated by a combination selected from the group consisting of: NspA, -Hsf, Hap, PorB, 〇MP85 'PilQ, PldA, FrpB, TbpA 'TbpB, FrpA, FrpC, LbpA, LbpB, FhaB, HasR, lipo〇2, Tbp2 (lip〇28), MltA (lip〇30) and ctrA. 11. A large blister preparation according to item 2 of the scope of the patent application, which can be prepared using at least method h), wherein one or more genes are inversely regulated by a combination selected from the group consisting of galE, siaA, siaB, siaC , siaD, ctrA, ctrB, ctrC and ctrD. 12. A large water mark preparation according to the scope of claims 1, 1 or 2, for use in the preparation of a composition for immunizing a human host to antagonize a disease caused by a Neisseria meningitidis infection. 13. An immunogenic composition comprising a large aqueous cancer preparation according to item i or 2 of the patent application and a pharmaceutically acceptable excipient. 14. A meningococcal immunogenic composition comprising a large blister preparation according to claim 1, 2, 1 or ,, and one or more selected from the group consisting of serotypes A, C, Y or W A simple or conjugated meningococcal capsular polysaccharide. 15. A meningitis immunogenic composition comprising a preparation according to the claims 1, 2, 10 or 11 of the patent application, a total of influenza bacillus capsular polysaccharide, and one or more simple Or conjugated pneumococcal capsules 65645-961123.doc _4. This paper scale applies to China National Standard (CNS) A4 specification (210X297 mm). A8 B8 C8 D8 1297731 VI. Patent application Fan Park 16· A modified N. meningitidis strain, whereby a large blister preparation of any one of claims 1, 2, 10 and 11 can be made. 17. A method of preparing an immunogenic composition comprising the step of producing the vaccine by a modified N. meningitidis strain according to item 15 of the patent application. 18. A genetically engineered large water sore preparation obtained from a modified N. meningitidis strain, characterized in that the preparation is obtained by the following processes: b) positively regulating large water sore preparation A method for the expression of a protective ο%? antigen. This method involves identifying the antigen, and the genetically manipulated strain introduces a stronger promoter sequence upstream of the gene encoding the antigen, such that the level of expression of the gene is less modified. Large blisters are high, and large blisters are made from the strain, and one or more of the forward-control genes are selected from the following combinations: NspA, Hsp-like, Hap' OMP85, PiiQ, PldA, TbpA, TbpB, FrpA, FrpC, LbpA, LbpB, FhaB, HasR, lipo02, Tbp2 (lip〇28), MltA (lip〇30) and ctrA o 19. Genetically engineered large blister preparation according to claim 18 The preparation, wherein the preparation is further obtained by using one or more processes selected from the group consisting of: h) a method of reducing antigenic expression in a large blister preparation, the antigen sharing structural similarity with a human structure, and being human Lure 65645-961123.doc 1297731 In vivo immune response, which involves the preparation of a gene involved in antigen synthesis, genetically manipulating the strain to reduce or sever the performance of the gene 'and making large blisters from the strain; or 〇 a positively regulated large blister preparation A method for the protection of ο P antigen expression, the method comprising identifying the antigen, the genetically manipulated strain to introduce more than one set of genes encoding the antigen in the chromosome, and consisting of a heterogeneous, strong promoter sequence Control and make large blisters from the strain. • According to the large blister preparation of claim 18 or 19, the genetic manipulation steps of the methods b) 'h) and i) are carried out by homologous recombination, which occurs on the bacterial chromosome at least 3 nucleus The sequence of tannic acid, and the sequence of at least 30 nucleotides on the vector in the strain are transformed. • According to the large blister preparation of claim 20, the genetic manipulation step is carried out by double cross-homogeneous recombination, and the event occurs on the bacterial chromosome by at least 3 分隔 separated by the nuclear name: acid sequence, X′ Two sequences of nucleoside citrate, and two sequences of at least 30 nuclear citrates separated by a nucleotide nucleotide sequence 'Y on a vector transformed into a strain, wherein X and Y are involved in recombination exchange. 22. The large blister preparation according to claim 21, wherein the two nucleotide sequences are about the same length&apos; and wherein the carrier is a linear DNA molecule. 23. The large blister preparation according to claim 21, wherein the recombination of methods b) and h) occurs in the chromosomal region upstream of the start codon of the key gene, 1 〇 〇 〇 b p . 65645-961123.doc This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) · A8 B8 1297731 24. The large blister preparation according to claim 23, wherein in the method h), the nucleotide sequence X comprises a partial gene promoter region, and the nucleotide sequence Y comprises a weak promoter region, Or no promoter area. 25. The large blister preparation according to claim 23, wherein in method b), the nuclear phytic acid sequence Y comprises a strong promoter region for bacteria. 26. The large blister preparation according to claim 25, wherein the nucleotide sequence Y is embedded 2 to 6 bp upstream of the start code of the desired gene. 27. The large blister preparation according to claim 26, wherein the nucleotide sequence of the fentanic acid is embedded about 4 bp upstream of the start codon of the desired gene. 28. The large blister preparation according to the second aspect of the patent application, wherein the recombination of the method 匕) is carried out, such that the nucleotide sequence X comprises the coding sequence of the key gene portion. 29. The large water cancer preparation according to claim 21, wherein the recombination of method i) is carried out, such that the nucleotide sequence γ further comprises the number of sets of genes present in the sputum. 3. A large blister preparation according to claim 8 or 9 of the patent application, wherein the modified N. meningitidis is a serotype B strain. 31. The large water cancer preparation according to claim 3, which is obtainable by applying at least method b) and/or i), wherein one or more genes are positively regulated by a combination selected from the group consisting of: NspA, class-Hsf, Hap, OMP85, PilQ, PidA, TbpA, TbpB, FrpA, FrpC, LbpA, LbpB, FhaB 'HasR, lipo0 2, Tbp2 (lip〇28), MltA (lipo30) and ctrA. 32. The large blister preparation according to the third paragraph of the patent application, which can be applied by the application 65645-961123.doc ^ _______ _ , _ This paper scale applies the Chinese National Standard (CNS) A4 specification (210X297 public). 1297731 έ88 C8 _________D8__ VI. Patent-Scope Large-cancer preparation strains are prepared by one or more methods selected from the group consisting of porA, porB, PilC, TbpA, TbpB, LbpA, LbpB, Opa and 0PC. 33. The large water cancer preparation according to claim 3, which can be prepared by applying at least method h) wherein one or more of the genes are inversely regulated by a combination selected from the group consisting of galE, siaA, siaB, Siac, siaD, ctrA, ctrB, ctrC and ctrD. 34. Large blister preparation according to item i 8 or 申请9 of the patent application for the preparation of a composition for stimulating a human host against a disease caused by one or more of the following infections: Neisseria meningitidis , Neisseria gonorrhoeae 'H. lyrophilus, Moraxella catarrhalis, Pseudomonas aeruginosa, Chlamydia trachomatis and Chlamydia pneumoniae. 35. An immunogenic composition comprising a large blister preparation according to item 18 or 19 of the patent application and a medically acceptable excipient. 36. A meningococcal immunogenic composition comprising a large aquatic cancer preparation according to claim 30, and one or more simple or conjugated ones selected from the group consisting of serotypes A 'C, Y or W Meningococcal capsular polysaccharide. 37. A meningitis immunogenic composition comprising a preparation according to claim 30 of the patent application, a conjugated H. influenzae capsular polysaccharide, and one or more simple or conjugated pneumococci Capsular polysaccharide. 38. A modified N. meningitidis strain, whereby a large blister preparation of the patent application No. 18 or 19 can be made. 39. A method of preparing an immunogenic composition comprising producing the set 65645-961123.doc -8 by a modified N. meningitidis strain according to claim 38 of the scope of the patent application. China National Standard (CNS) A4 specification (210 X 297 mm) A8 B8 C8 D8 1297731, the procedure for applying for a patent range. 40. A modified N. meningitidis strain, characterized in that the strain is obtained by the following process: d) a method of removing LPS lipid A partial toxin in the strain, the method comprising: identifying The lipid a part of the LPS is a toxic gene, the genetically manipulated strain is used to reduce or sever the performance of the gene; and h) the strain is genetically manipulated to have no capsular polysaccharide. 41. The modified N. meningitidis strain according to claim 40 of the patent application wherein the gene has the nucleotide sequence of SEQ ID NO: 81 42I 42. According to the scope of claim 4 or 4 A modified N. meningitidis strain in which the gene is inversely controlled by point mutation or deletion. 43. A modified N. meningitidis strain according to claim 40 or 41, wherein the open reading zone or the promoter of the 5 gene is deleted or all. 44. 45. A modified N. meningitidis 2-ball strain according to Section 4 or 4 of the patent application, wherein the A-base of the biosynthesis or export of the capsular polysaccharide is mutated by the gene The control area or coding area is not activated. According to the patent application scope 40 or 41 modified meningitis naphtha = cocci, in which the base of the biosynthesis or output encoding the capsular polysaccharide T' is controlled by point mutation or deletion . &quot; 46·Modified meningitis Neisser according to the scope of patent application No. 40 or 41 65645-961123.doc This paper ruler is used in the grid (2ι〇χ297 mm)· 1297731 A strain of the bacterium, wherein one or more of the genes in method h) is inversely controlled by a gene selected from the group consisting of g E siaA, siaB, siaC, siaD, ctrA, ctrB, c t r C and c t r D . 47. A modified meningococcal strain of B. cerevisiae according to Article 4 or 41 of the patent application, which is a serotype strain. Human "48. Two genetically engineered large blister preparations prepared from a modified N. meningitidis strain according to item 40 or 41 of the patent application. 49. A genetically engineered large blister preparation for the preparation of a composition for modulating a disease caused by a Neisseria meningitidis infection for immunizing a human host. 5〇, an immunogenic composition, A genetically engineered large blister preparation and a medically acceptable licking agent according to claim 48 of the scope of the patent application. 51. The immunogenic composition according to claim 5, further comprising one or more A simple or conjugated meningococcal capsular polysaccharide selected from the group consisting of serotypes A, c, sputum or W. 52. The immunogenic composition according to claim 5 of the scope of the patent application, further comprising a conjugated H. influenzae capsular polysaccharide, and one or more simple or conjugated pneumococcal capsular polysaccharides. 53. An immunogenic composition for antagonizing N. meningitidis, according to the scope of the patent application 4 0 or 4 1 Produced by the Neisseria meningitidis strain of modifications. 54. The scope of the patent application of 3 5 immune immunogenic composition into ~ 65645-961123.doc, 1〇. This paper scale applies to the Chinese National Standard (CNS) A4 specification (210X 297 mm). 1297731 The step comprises one or more simple or shared meningococcal capsular polysaccharides selected from the group consisting of serotypes A, c, Y*w. The immunogenic composition according to item 53 of the patent application further comprises a conjugated H. influenzae capsular polysaccharide, and one or more simple or conjugated pneumococcal capsular polysaccharides. 56. A method of preparing an immunogenic composition for antagonizing Neisseria meningitidis comprising the step of producing the vaccine by a modified N. meningitidis strain according to item 4 or 4 of the scope of the patent application . 57. A modified N. meningitidis strain according to the fourth or fourth aspect of the patent application, for use in the preparation of a combination for antagonizing a disease caused by a Neisseria meningitidis infection for immunizing a human host Things. 65645-961123.doc _ This paper scale applies to China National Standard (CNS) A4 specification (210X297 mm)