TW201035317A - Recombinant HA influenza virus-like particles and vaccine composition thereof - Google Patents

Abstract

The present invention relates to recombinant HA influenza virus-like particles (VLPs) derived from Baculovirus expression system, and their use in preparing protective influenza vaccine composition.

Description

201035317 VI. Description of the Invention: [Technical Field] The present invention relates to the manufacture of influenza virus recombinant HA-like virus particles (VLP) by a baculovirus expression system, and a preparation thereof for preparing a chicken-protective influenza VLP vaccine Composition. [Prior Art] Influenza virus (F) is classified as a positive mucinous virus family (βτ幼) in terms of virus classification, according to the difference between its internal ribonucleoprotein (NP) and matrix protein (Μ). Divided into sputum, sputum and C virus. Among them, sputum and influenza C viruses are mainly infected, and influenza A viruses have a wide range of hosts in nature, including humans, pigs, horses, whales, seals, wild birds and poultry. At present, the smell of bird flu is "influenza A". The genome inside the influenza virus is composed of a number of negative-negative RNA fragments. The genomes of influenza A and B viruses are composed of 8 fragments, while the influenza C virus contains only 7 fragments. The eight-stage gene of influenza A virus can be expressed in dysentery (10), including nuclear protein (rib_leQpr〇te^ NP), three polymerase protein units (PA, PB1 and pB2), and two matrix proteins (M1 and M2). 2 non-structural proteins (NS1 and NS2), 2 external glycoproteins, including hemagglutinin (M) and neuron (neuraminidase, upper. > II virus particles all have lipids in appearance Envelopes... Lihua (6) is obtained from the host cell membrane during the course of the disease. The surface of the envelope is embedded with the most amount and the number of M2 proteins is small. The inner surface of the envelope is related to it. The function and binding of the viral particles to the host cell host cell promotes fusion of the envelope with the cell membrane (the function of the membrane to release the viral genome's NA protein is to help the newly formed A = if to detach from the cell surface and thereby infect adjacent cells. ^ lectin (HA) and neuraminidase (10)) (m,), 16 subtypes of HA (H1 side) and 9 subtypes of Μ subtype are known to be only subtypes of Η1-Η3 and N1-1V2 Can directly feel 201035317 dyed people's and spread through A and people. These subtypes of viruses do not usually cause disease in wild waterfowl, but the mortality rate of poultry after infection is extremely high, so the annual economic losses caused by the poultry industry are the first to erupt in Hong Kong since 1997. The H5N1 flu virus infects humans, and has since spread disasters in Asia and spread rapidly to mainland China, including the Middle East, Europe, and even Africa, where there are few flu epidemics, and so far (June 31, 1996, WHO) Statistics: 319 people were infected, 192 people died, and the mortality rate was as high as 60%. In order to effectively prevent the occurrence of influenza, the most important measure is to apply the 'flu vaccine. The current influenza vaccine is to use chicken embryo culture virus. Vaccines that have been deactivated. Every year, the World Health Organization and the United States Centers for Disease Control will recommend vaccine components based on data from previous years in the world, and speculate on the types of viruses that may be prevalent the following year. In the past few years, because of the simultaneous prevalence of H1N1 and H3N2 viruses, the ingredients of the human influenza vaccine include both types of A. Influenza virus and a type B influenza virus. However, this vaccine does not provide sufficient protection against the currently circulating strains of the H5 and H7 subtype influenza viruses. In addition, due to the southern pathogenic H5 and H7 viruses will kill Dead chicken embryos, so it is impossible to achieve large-scale production by direct inoculation to chicken embryo eggs. The isolated high-pathogenic wild virus strain must first be converted into a weak virus strain, which will inevitably lead to the complexity of the vaccine process. The glycoprotein on the surface of the influenza virus particle determines the pathogenicity of the virus itself. ° The functional prion protein is structurally in the form of a trimer. The HA protein can be combined with the surface of the host upper respiratory tract epithelial cell. The binding of the terminal sialic acid on the protein molecule allows the virus particles to enter the cell using endocytosis. The HA protein promotes membrane fusion in the acidic environment of the endosome (pH 4~5). The genome of the virus is released. The HA protein can be cleaved into HA1 and HA2 subunits in the cell, and the two subunits are linked together by a disulfide bond. The highly pathogenic HA protein cleavage site carries multiple continuous basic amino acids. The sequence (-PQRERRRKKfVGL-), therefore, needs to be changed to the amino acid sequence of the attenuated strain (PQ----RETR/G) and NA after the vaccine preparation, and then with the other 6 from pR8 disease 201035317 ^ strain (10) _ Gu, _ The gene fragment is made into the recombinant virus lrrriLatirus), and then the reverse gene method is used to prepare the attenuated vaccine strain. The vaccine strain prepared by this method is suitable as a prototype for the vaccine. Strain or mock vaccine strain (Mock-up vaccine). In 1997, when Hong Kong broke out, Japan and the boots were exposed to the disease. The N1 virulent strain isolated from the diseased duck was made into a weakly toxic influenza vaccine using the reverse genetic method. As a result, the test animals were exposed to the blood for 2 times. There is an efficient response to the antibody. (National Institutes of Health Newsletter No. 213 2007-08-10). ◎ E. coli U α ^ / ;) is currently the most widely used protein expression system, with the advantages of rapid growth, south yield and low cost. However, since Escherichia coli is a prokaryote that cannot be glycated (glyc〇sylati〇n), it is impossible to produce st eggs, and products often form insoluble inclusion bodies (inclusi〇n body), so that the protein itself does not have Biologically active or only very low biological activity. Conversely, the mammalian cell system has complex saccharification and complete post-translational modification. The produced protein is less likely to form inclusion bodies, and has similar properties to natural proteins, and most proteins are secreted. It is easy to recover in the culture solution. The shortcomings are long development time, low yield, high base, and low mass production, so at present, apart from some large biotech companies (such as Amgen,

In addition to the fact that Genentech, the Genetics Institute, etc. are mastering key technologies and being able to industrialize, other industries are still in the rush to catch up. Compared with the former two, the rod-shaped virus performance system is an emerging technology in recent years, which has the advantages of combining the first two systems. Baculovirus is a rod-shaped insect virus containing a double-stranded DM gene. Because it has several extremely strong promoters (pl〇, polyhedrin promoter), it was developed into an effective foreign protein production in the early 1980s. tool. The study of influenza VLPs in the baculovirus expression system is first

Published by Latham and Galarza (t. Latham and JM Galarza, 2001 (Latham, T., and JM Galarza. 2001. Formation of wild-type and chimeric influenza virus-like particles following simultaneous expression of only four structural proteins. J Virol 75 :6154-65.) > The sputum, NA, Ml and M2 genes of influenza A/Udorn/72 (H3N2) were constructed on the same baculovirus vector in 201035317 and expressed in Sf-9 insect cells after infection. The culture supernatant contains influenza VLPs' and was confirmed by penetrating electron microscopy. This report confirms that influenza VLPs can be formed by the co-expression of HA ' NA, Ml and M2 genes, and the resulting VLPs are Polymorphism is very similar to real virus, with a diameter of between 80 and 120 nm. Subsequently, Pushk〇 et al. (p. pushko et al., 2005 (Pushko, P., TM Tumpey, F. Bu, J. Knell, R.). Robinson, and G Smith. 2005. Influenza virus-like particles comprised of the HA, NA, . and Ml proteins of H9N2 flu virus protective protective responses in BALB/c mice. Vaccine 23:5751-9·)) will also flow The sputum, NA and M1 genes of the virus A/Hong Kong/1073/99 (H9N2) were constructed on baculovirus scorpion venom and expressed in Sf-9 cells, which was also confirmed in the supernatant of the medium. The presence and appearance of H9N2 VLPs were not significantly different from those published by the author Qin. In addition, Pushko et al. also vaccinated BALB/c mice with a VLP vaccine of 1 ,, after 2 subcutaneous vaccinations ( On Days 与 and Day 28) and after challenge, the results showed that influenza VLPs not only elicited highly effective HI antibodies (128-256 times) in mice, but also showed protection effects after challenge. The lung virus and nasal secretions of the mice inoculated with the VLP vaccine were significantly lower than those detected in the control group, indicating that the influenza virus could not be effectively replicated in the vaccinated mice. This report confirms the potential of developing a human fluent vaccine with a baculovirus expression system. Similar experiments can also be found in the study by Bright et al. (R. A.

Bright et al., 2007) (Bright, R. A., D. M. Carter, S. Daniluk, F. R.

Toapanta, A. Ahmad, V. Gavrilov, M. Massare, P. Pushko, N. Mytle, T. Towe, G. Smith, TM Ross. 2007. Vaccine 25: 3871-3878.), they are in Sf-9 cells The CCP showed the HA, ΝΑ and M1 genes of influenza virus A/Fujian/4ii/2〇〇2 (H3N2) and quantified the amount of VLPs based on the concentration of ha protein in the sample. Bright et al. used different amounts of VLPs (24ng-3pg) with deactivated H3N2 influenza virus (600ng) and purified recombinant HA protein (600ng) to immunize BALC/c mice and Fitch ferret to compare their effects. . The results showed that the titer of HI antibody in mice was related to the amount of VLps received. The higher the dose, the higher the HI antibody was induced. It can be seen that the use of influenza VLPs as a vaccine can lead to 6 201035317 == I can get a shirt VLPs * i^t^rnrn^ Christine "He is engaged in the study of insects and secrets, and only a few prove that HA is in the sputum! The envelope of human fl-virus is released in the process of infection.

SUMMARY OF THE INVENTION The present invention provides a recombinant virus-like age (VLP) comprising an influenza virus glycoprotein, wherein the venom has a diameter between 80 and 150 nm. The "invented by ί"-specific embodiment of the recombinant attenuated granules is produced by expressing the HA protein gene alone in a baculovirus expression system. In another embodiment of the invention, the recombinant virus-like particle is produced by expressing the white gene and the Ml egg from the gene in the secret of the virus. In yet another embodiment, the recombinant virus-like particle is produced by expressing the HA protein gene and the M1 protein and the NA protein gene in a baculovirus expression. In another aspect, the present invention provides an influenza VLP vaccine composition comprising an influenza virus recombinant HA-like virus particle (VLp) prepared by a baculovirus expression system as an effective component, and a pharmaceutically acceptable carrier Agent. In one embodiment, the influenza virus is an influenza virus H7 subtype virus. In a specific aspect of the present invention, the influenza virus recombinant HA-like virus particle (VLP) is obtained by expressing HA egg from the gene paste at the age of the baculovirus. In another aspect of the invention, the influenza virus recombinant HA-like virus particle (VLP) further comprises an Ml protein. In yet another embodiment, the influenza virus recombinant HA-like virus particle (VLP) further comprises a prion protein and a NA protein. 7 Other characteristics and advantages of 201035317 will be described in detail from the lower touch and several specific embodiments, and are also apparent from the scope of the attached patent application. [Embodiment]

Hi stores the domain virus 11 group HA-like disease from the baculovirus table m. According to the method of the present invention, it is first discovered that the baculovirus expression system =' HA protein can be assembled into VLPs by itself, and does not require the M1 protein or the interaction of 趴 with NA t. And by the chicken immunoassay and HI antibody titer analysis, the sero-recombinant HA-like virus particles of this sacred month can effectively induce the production of enough sputum with the mouth! The antibody 'Lin Guan Qian Xian reduced the amount of fruit into a fruit. Hereinafter, the present invention will be described in detail with reference to specific embodiments. These specific examples I are provided by the explanation of the invention and are not intended to limit the invention. The present invention is intended to embrace such modifications and variations and modifications. Example 1 Preparation and characterization of influenza virus recombinant HA-like virus particles (VLP) a·Golden and plastid construction and baculovirus acquisition The HA gene of influenza virus H7N1 has been previously constructed on pCRn-TOPO plastids. (pCRjI-H7), provided by Zhang Bojun, teacher of the Veterinary Microbiology Institute of Zhongxing University. In order to construct a vector for the baculovirus expression system to obtain a recombinant baculovirus for expression, the fragment containing the full-length HA gene and the deleted ejjA7h sheet will be first added (removing the transmembrane and cytoplasmic domain sequences at the C-terminus, increasing 6xHis) The tag sequence was constructed on the pBlueBac4 vector to obtain two recombinant plasmids, PBB4-H7 and pBB4-eHA7h, respectively. The recombinant baculovirus, vBac-H7 and vBac-eHA7h were obtained by transfecting the above two recombinant plasmids into Sf9 worm cells using Invitrogen's Bac-N-Blue transfection kit. Since previous studies have confirmed that influenza-like virus particles can be produced in insect cells by means of protein co-expression, in order to investigate and compare the immune effects caused by the different components of the virus-like particles and recombinant HA protein in the chicken, We used the Bac-to-Bac system (Invitrogen) to construct two recombinants for co-expressing infections. 201035317 VMH # νΜΜΝ 'The insertion gene maps of these constructs such as = BaC|Blue system co-transfection (transfer The procedure is as follows: 4 _ and plastid pBM_ ^ gm 〇 5 μδ AcNPV DNA ^ 25 μ1 iipofectin ml ^ ^ base offense, let stand for 15 minutes. Inoculate M () 6 Sf-9 cells at 6 (10) A little G vein, S medium wash cells 2 *. Mix the above ΐ for at least 5 hours. Replace the previous mixture with 2 ^ 1%% J + face-FH medium, and _ culture in the form of τ. Purification of recombinant baculovirus purification: 〇? : 9 cells were diluted with disease - (4), and the cells were covered with medium containing 0.5% ginga and 50 pg/ml X-gal, placed on 28 〇c culture for 7 days. Pick blue plaque infection Sf-9 fine 'Collect the supernatant after infection, centrifuge to remove cell debris and store at 4 °C' This is the first generation virus mother liquor. Repeat with the first generation virus mother liquor. Infect Sf_9 cells to amplify the virus titer to 109 pfu/ml or more. Briefly describe the Bac-to-Bac system and virus screening process as follows: • Transform the recombinant plastid constructed in PhstBacl into a special C (^DH1〇Bac competent cell/containing 50 pg/ml kanamycin, 10 pg/ml Screening of tetracyclin, 7 pg/ml gentamycin, 40 pg/ml IPTG, and 100 pg/ml X-gal. Q The selected white transformed strain was cultured in a small amount of LB' to extract its bacmid. Take 1 recombinant bacmid DNA, 20 μΐ Cellfectin and 1 ml Grace's medium were mixed evenly. Allow to stand at room temperature for 15 minutes. Inoculate ΐχΐ〇6 Sf-9 cells in a 6 cm dish. Wash the cells twice with a little Grace's medium. Add the above mixture to the culture. In the dish, culture at 28 T for at least 5 hours. Replace the previous mixture with 2 ml of TNM-FH medium containing 1% fetal bovine serum and continue to culture at 28 ° C. After 5 days, collect the supernatant and centrifuge to remove the cells. Residue is stored in 4T, this is the first Virus stock. In the first generation of virus stock Sf-9 cells infected with repeated viral titers to enlarge 109 pfu / ml or more backup. 201035317 b. The performance of the recombinant protein in the baculovirus expression system. The cells were first cultured in a medium containing 10 ml of ESF921, such as S Γ. The initial cell density was about 5x105 eel ls/ml, at 250 rpm (TKS orbital shaking Under the conditions of incubator). Gradually expand to a total volume of culture 3 _ when the cell density is increased to 丨 & with 6 of the aforementioned recombinant baculovirus infection, after the infection of 72 1 heart (3 just ^ 1G minutes), the cell is placed in the ί 1 ί is followed by purification of the protein. The supernatant was subjected to purification and analysis of influenza VLPs. 〇 蛋 蛋 蛋 Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi In the case of carrying a single-gene virus vBac-H7 infection, Hi-5' has a HA expression of 9.. μ§/ιη1; and the total expression of Hi-5 is infected with the Ml gene virus vBac-MH. Increased to 69. 8 ± 2. 9 pg / ml, a total of HA, sputum and Ml gene vBac-HMN infection Ηι-5, the HA performance was not significantly different from the infection of vBac-H7, about 8 2 Mg/nd. As shown in the results of Figure 2B, Hi_5 cells were infected with vBac_H7, about 2.0% of HA protein was secreted into the medium; when infected with vBac_MH, M and m proteins were expressed in the cells, and the proportion of HA protein was released. A slight increase to & 1 ± 〇. 7%; with vBac-HMN infection 'Μ, NA and Ml protein co-expression, the proportion of HA protein release significantly increased to 7.5 ± 2.5%. c. Purification of influenza VLPs The supernatant (300 ml) after infection of Hi-5 cells was filtered with a 〇·45 _ filter membrane (Pall corp·), and then subjected to a low-temperature concentrator (Labscale^ TFF) at 4 ° C. System, Millipore) The filtrate is a 5〇〇kDa-concentrated membrane (PelliC0nXL,

Biomax 500, Millipore) is concentrated to approximately 60 mi. The concentrate was subjected to ultracentrifugation (OptimaTM XL-80K 'SW41i rotor, Beckman), and centrifuged at 27,000 rpm ' 4 ° C for 3 hours. The pellet after centrifugation was dissolved back into 2 mi of TNE buffer (10 mM Tris [ρΗ8·0], 100 mM NaCl, 1 mM EDTA). The remelted sample 201035317 was added to the top of a 20%-60% (20-40-50-60) sucrose gradient solution, and subjected to ultra-high speed centrifugation at a temperature of 27 000 rpm ' 4 ° C for 16 hours. A fraction is charged for each sister from top to bottom. The M titer of each layer was determined by a hemagglutination test, and the density of each layer of the curtain sugar solution was measured by a density detector (Milton Roy Company), and the distribution of the protein was detected by Western blotting. 3 is shown. Fig. 3A shows the results of HA alone. The HA protein signal is mainly distributed in the layer 4~7, and the strongest signal appears in the layer 4'. The HA titer of this layer is 1: 512, and the sugar density is equivalent to 1.101 g/cm3. Figure 3B shows the results of co-expression of HA and M1 proteins. The HA protein signal is mainly distributed in the layered 5~7'Ml protein signal mainly distributed in layer 5, the strongest signals of both appear in fraction 5, HA titer of this layer For 1: 2048, the sucrose density is equivalent to L 124 g/cm3. Figure 3C shows the results of a total of μ, like " and M1 protein. The HA protein signal is mainly distributed in the layer 4~9, the ΝΑ protein signal is mainly distributed in the layer 5 and 6, and the Ml protein signal is mainly distributed in the layer 4~ 9, each protein is the strongest, the signal appears in layer 5, the HA titer of this layer is 1: 2048, the curtain sugar density phase * when L 146 is (10) 3. Figure 3B and the diagram demonstrate that when insect cells are infected with ^Bac_Mf or vBac-HMN, 'influenza proteins (HA and M1 proteins or HA, NA and Ml proteins) are co-released out of the cell and can be centrifuged in a common sucrose gradient. Purification of sputum by stratification to this result suggests that HA and M1 proteins or HA, Μ and Ml proteins can be assembled into VLPs. In addition, when cells are infected with vBac-H, HA is also released out of the cell, and can also be purified in a layer of sucrose density equivalent to KVLPs. This result suggests that HA proteins can also be assembled into VLPs when they are expressed alone. . d. Characterization of influenza virus recombinant HA-like virus particles (VLP) From the stratification collected after sucrose gradient centrifugation, we can determine the results of HA alone and the co-expression of HA with Ml and/or NA proteins, due to these differences Large proteins are distributed in sucrose solution of the same density after centrifugation, indicating that the possibility of assembly into LPs is high. To verify this, we concentrated the sucrose gradient sample and observed it under a transmission electron microscope. The results are shown in Figure 4-6. I 201035317 ^ observed influenza VLPs, the shape of the polymorphic granules 'mostly round or elliptical true flu disease at about 80 ~ 150 nm door, cypress b ~ r, round $ ' > number For irregular shape 'diameter

^ λ 0 and the surface protein 屮fHA can be clearly seen

Protein) and membranous structure. Amazing beta = 彳 = morpha protein dog (HA requires Ml protein "with ° ΝΑ = total egg with = alone assembled into VLPs, not 重组 recombinant HA-like virus particles (VLP) immunoassay and red blood Agglutination inhibition (HI) antibody titer analysis The immune effects of recombinant M protein and influenza-like virus particles produced by ΐ , 以 , , , , , , 以 以 免疫 免疫 免疫 免疫 免疫 免疫 免疫 免疫 免疫 免疫 免疫 免疫 免疫 免疫 免疫 免疫 免疫 免疫 免疫 免疫 免疫 免疫 免疫 免疫 sp sp sp sp sp The contained protein is a quantitative standard, and the inoculated chickens are divided into 8 groups, including H_VLP (2 pg) and H_VLp (〇〇MH-VLP (2 μ§) ^ MH-VLP (0.2 μ§) ^ HMN-VLP (2 μβ) > HMN-VLP (〇·2 μ2), eHA7h (100?) and ·, the name represents the type of antigen to be inoculated and the quail egg contained in the vaccine cap. Immunization due to domain vaccine Anti-hybrid is poor, so each chicken is vaccinated twice, and adjuvant is added to the vaccine to increase the effect of the immune response. The first injection uses complete jrreund's adjuvant, and the second immunization after two weeks is not used. Complete Freund's adjuvant. The blood serum agglutination inhibition test ELISA, respectively, measured HI antibody titer and anti-HA antibody titer in serum, and the results obtained are shown in Figure 7. HI antibody titer is an important indicator for judging the presence of protective antibodies in serum and vaccine effect. The test was carried out with a 96-well V-type microassay plate. Pack 25 μΐ 〇. 85% NaCl to the parent hole of the V-plate and add 25 μ in the first well _ test serum, from the first The wells were started with two consecutive serial dilutions, diluted to the last well and discarded for 25 μΐ. Add the diluted virus solution containing 8 agglutination units (HAU) to each well and pat the plate to mix evenly and place at room temperature. Minutes. Add 50 μΐ 0.9% chicken red blood cell suspension to each well and pat the plate to mix evenly. After standing for about 60 minutes at room temperature, the blood clot agglutination inhibition is recorded and recorded. Tilt the plate, such as 12 201035317, Zhu Cheng tears down and the rate of flow down is the same as that of the blood cell control group, indicating that the blood cell 7G is fully settled and the agglutination is inhibited. The highest dilution factor of the complete sedimentation of the blood cells, which is the serum HI antibody titer. antibody The potency of 241 〇 became the lowest protection threshold, meaning that when the serum was diluted 16-fold, the function of completely inhibiting erythrocyte agglutination was still achieved in the standard hemagglutination inhibition test. The results are shown in Table 1 below and Figure 7.

〇〇 Group injection volume 2 weeks before the 3rd week 4th week 5th week 6th week 7th week 8th week 12th week 16th week HI antibody titer IX takes the average value of 1 〇g2 ^矣#. One of the chickens in this group died before the first vaccination. The left chicken was not ※. The chickens in this group received the third immunization at the 5th week: no agglutination inhibition was detected. X: Not tested. As can be seen from Table 1 and Figure 7, in the group receiving the 2 virus-like particle vaccine, all the chickens were within 5 weeks after the first immunization (week 2), at 5·5±0·6 (H- VLP), 5.8 ± 1 · 1 (MH-VLP) and 5. 5 ± 〇. 7 (HMN-VLP). After the first immunization, all chickens receiving 2 pg of virus-like granule vaccine were guilty of 13 201035317 antibody titers reaching the highest in a week or two, with the highest value of each group being 8.0 ± 0.4 (H-VLP) 9.3±1.8 (ΜΗ~νΪ) 7 5+3 (HMN-VLP). In addition, 'when _ her county-age vaccine towel Jin: · for the original 1 / Η) (0. 2 咫) when 'all the difficulties are only in the second no detective _ greater than the value of the serum value of the standard shed, but in the first The second week (week 4) 'All serum HI antibody powers are ^ ^ log2^i^ 8.3 ± 1.8 (H-VLP), 4 m and 6 · _LP) 'which accepts 〇. 2 heart M=P Anti-two-force price - in the group that exceeds the standard value and accepts (iv) hi (4.8±〇.4 jeM7h), even if the antigen amount is as high as (10), the HI antibody is still I method ίίΊΪίίΪΐ When we continue to be at the 5th week, the first immunization's serum can be measured after the 6th week to have a standard threshold of HI antibody power (week 6_8). It is worthwhile—================================================================================================== There was no significant reduction in the middle, even at 98 days after 5 a* hall-V / 3 immunization), at least 32 times the force price was measured, and the effective Ηι force induced by 3 not = gI rf granule vaccine maintained at least 98. Day reorganization I white miscellaneous sales ί l.if virus particle vaccine and accept 100 pgeHA7h she fights > Lu Chu ^9 'the blood of the month can maintain a protective power price for a shorter period of time, two weeks The price of the time is reduced below the standard. It can be seen that 2 ΐϊΐΓΐίϊΐί苗 can cause a more effective immune response in chickens for a longer period of time. It can be seen that in the case that the flow particle is superior to the weight, the virus-like particles are better than the recombinant protein in terms of the amount of effect or cost, and it is more suitable as the first choice for the new-generation influenza vaccine. 201035317 Other specific states 檨 Yes, all features of iz can be combined in any combination. Replaced by the alternative feature of ash. The sect of the sect can be determined by the same, equal or similar purpose, and the features of the age are described from the foregoing, the characteristics of the heat, and the skilled person can easily determine the invention at an unaged age. The basis of the other aspects is also included in the scope of the patent.

15 201035317 [Simplified illustration of the schema] Figure 1 is a map of the inserted gene fragment of the recombinant baculovirus co-expressing infection. All recombinant baculovirus constructs only showed homozygous recombination (h〇m〇i〇g〇us recombination) regions, and the related restriction enzyme recognition sequences were uniformly labeled on the single gene expression virus vBac-H. Other markers: Tn7R/7L, homogenous recombination sequence; Gm' gentamycin resistance gene; PolH, polyhedrin promoter sequence; P(A), polyadenylation sequence. Figure 2 shows the amount of HA protein in the co-expressed Hi-5 cells (A) and the ratio of HA and M1 proteins released to the extracellular when the co-expressed infection (B) ea is Hi-5 cells in a rod shape HA protein expression at the time of virus vBac-H, vBac-MH, vBac-HMN infection, C) All quantitative samples were taken from cells after 72 hours of infection with moi=5. Above the B-picture, the intracellular and extracellular HA and Ml proteins were analyzed by Western blot, and the ratio of the quantified HA and Ml proteins released to the extracellular is shown below the b-picture. Figure 3 is a sucrose gradient (20 to 60%) for centrifugation analysis of infected Hi-5 cell culture supernatant. Among them, 300 ml of Hi-5 cell solution was infected with m〇i=5 recombinant baculovirus (A) vBac:H, (B) vBac-MH, (C) vBac-HMN, and cell fluid was collected 72 hours after infection. The cells and the culture supernatant were separated after centrifugation. The supernatant fraction was treated with the influenza VLPs purification method (Example i.c) and analyzed by Western blot and hemagglutination assay. The bottom of the figure shows the q sucrose density (g/cm3) and the HA force price measured for each layer. Figure 4 is a transmission electron microscope image of the MH-VLP. The images come from the same product and are magnified 1 million times in different fields of view. The bar in each figure is equal to 1〇〇 nm ° . Figure 5 is a transmission electron microscope image of the HMN-VLP. The images were taken from the same sample and were taken at a magnification of 10,000 times in different fields of view. Bar 100 nm in each figure. Figure 6 is a transmission electron microscope image of the H-VLP. The images are from the same product, and they are obtained by zooming in and out of the field for 10,000 times. (8) in each figure is equal to nm〇, and Figure 7 shows immunization with recombinant subunit vaccine and virus-like particle vaccine sp{?chicken 201035317 white, i times per it two. The /f group subunit vaccine is two eM7h eggs = r: BS). ' #": One of them died before the first injection. The titer of the antibody was calculated as the mean ± standard error of each group (standard deviati〇n). Preiramune serum was collected before the first immunization; week 2 The serum is collected before the second immunization. 'The blood is collected once a week, and every 4 weeks after the 8th week, the blood is collected once. 0 [Main component symbol description]

17

Claims (1)

201035317 VII. Patent application scope: 1. Influenza recombinant virus-like particle (VLP) containing influenza virus glycoprotein HA protein, wherein the recombinant virus-like particle has a diameter of between 8〇15〇(10). 2. The influenza recombinant virus-like particle (VLP) according to claim 1 of the patent application, which consists solely of the influenza virus glycoprotein HA protein. 3. Influenza recombinant virus-like particles (VLP) according to item 2 of the scope of the patent application, wherein f is obtained by expressing quail eggs from the gene alone in a rod (four) toxicity expression system. 4' According to the patent scope of claim 1, the influenza recombinant virus-like particle (VLP), which is produced by the 〇 ΗΑ ΗΑ protein-based transgenic M1 protein gene in a baculovirus expression/system. 5. According to the patent application scope! The influenza recombination-like virus particle (10), the pottery protein gene and the M1 protein and the NA protein gene are expressed together in a rod-like poisoning expression system. 6. 2ΐΐνΐί vaccine composition 'which consists of a baculovirus expression system; = virus-like particles (10)) as an effective component, and a system for making a stream; a stream of vaccines, wherein the stream 8·, according to Apply for a patent to turn the seventh VLP vaccine composition of the influenza virus H7 subtype virus. According to the scope of the patent application, the 8th V VLP vaccine composition 'where the influenza virus enters the subtype influenza virus. 7匕左共他冋9· f : Please refer to the patent scope of item 6 of the influenza VLp vaccine composition, susceptibility, and recombinant virulence virus particles (VLP) by performing the performance in the ovarian baculovirus expression system. Got it. "Protein gene alone in A = Influenza VLP vaccine composition of claim 6: Recombinant virus-like particles (VLP) are composed only of influenza virus glycoprotein U. According to the influenza VLP vaccine of the application patent item 6 The stream, wherein the stream 18 201035317 ίίίΐί HA-like virus particles (VLP) further contains prion protein. 12. The influenza VLp vaccine composition of claim 6, wherein the fluent sputum recombinant HA-like virus particle (VLp) further comprises m protein and na^ white. 13. The influenza VLp vaccine composition according to claim 13 of the patent application, wherein the A protein comprises influenza VLP vaccines from different subtypes of influenza virus to combine chimeric VLPs. 14. H2 patent scope item 6 The flu-like vaccine composition is used to protect chickens from flu = more.中 ❹ 〇 19