CN1245512C

CN1245512C - Vaccines containing recombinant pilin against i cneisseria gonorrhoeae (or i cneisseria meningitidis)

Info

Publication number: CN1245512C
Application number: CNB998056014A
Authority: CN
Inventors: T·N·梅特卡夫三世; R·J·扎古尔斯基; P·奥依
Original assignee: Wyeth Holdings LLC
Current assignee: Wyeth Holdings LLC; Wyeth LLC
Priority date: 1998-04-29
Filing date: 1999-04-29
Publication date: 2006-03-15
Anticipated expiration: 2019-04-29
Also published as: WO1999055875A2; KR20060118629A; JP2002527041A; BR9910005A; KR20060118628A; EP1073748A2; AU3968599A; WO1999055875A3; CA2325055A1; CN1298446A; IL139311A0; KR20060118630A; KR20010043105A; AU2003204738B2

Abstract

The pilE genes of each of Neisseria gonorrhoeae and Neisseria meningitidis are cloned and their corresponding recombinant pilin proteins are expressed. In addition, a chimeric pilE gene is constructed in which the region of the pilE gene of Neisseria meningitidis class I encoding the amino-terminal region of the pilin protein is replaced by the corresponding region of the pilE gene of Neisseria gonorrhoeae. The recombinant meningococcal chimeric class I pilin protein is expressed at higher levels than the pilin protein expressed by the full-length pilE gene of Neisseria meningitidis. Furthermore, a chimeric pilE gene is constructed in which the region of the pilE gene of Neisseria meningitidis class II encoding the carboxy-terminal region of the pilin protein is replaced by the corresponding region of the pilE gene of Neisseria gonorrhoeae. The recombinant pilin proteins are used in vaccines to protect against disease caused by Neisseria gonorrhoeae or Neisseria meningitidis.

Description

Contain the anti-Diplococcus gonorrhoeae of recombinant pilin or the vaccine of Neisseria meningitidis

Invention field

The present invention relates to the disease that the employing recombinant pilin protects body prevention Diplococcus gonorrhoeae (Neisseria gonorrhoeae) or Neisseria meningitidis (Neisseria meningitidis) to cause in vaccine.

Background of invention

Diplococcus gonorrhoeae (N.gonorrhoeae) and Neisseria meningitidis (N.meningitidis) are Gram-negative coccus.Very close on Diplococcus gonorrhoeae and the Neisseria meningitidis gene, but difference is very big in the clinical manifestation of their associated diseases.Diplococcus gonorrhoeae is caused gonorrhoea, and Neisseria meningitidis causes meningococcal meningitis.These Neisserial bacteriums are present in the mucomembranous surface of body.

IV type pili is the non-flagellum hair shape structure on many gram negative bacteriums surface, these gram negative bacteriums comprise: the plethora artiodactyl shape bacterium of artiodactyl shape Pseudomonas (Bacteroides originally), erode the Aitken bacterium, denitrification (denitrogenation) Kingella, ox catarrhalis, lacuna catarrhalis, catarrhalis does not liquefy, Diplococcus gonorrhoeae, Neisseria meningitidis and Pseudomonas aeruginosa (Bibliography Entries 1,2).The bundle that the toxin of vibrio cholerae is regulated pili and enteropathogenic Escherichia coli altogether forms pili and shows similarity with IV type pili limited quantity, it is believed that it is remote relationship (1,2).For gonococcus and meningococcus, fimbriate bacterium is more much higher in various HE avidity than the bacterial adhesion of no pili, so think that pili is that the microorganism anchor in the virulence factor at mucosal infections position.

IV type pili is wide to be 5-7nM, and length is 5 μ M.The subunit of pilin is connected in series and forms long and thin polymer.For pathogenic eisseria, the pili outward appearance is natural homopolymer, forms pilin by the subunit of single structure.The molecular weight of pilin is 13,000-22,000 dalton (1,2,3).

The natural bacterial outer membrane that is assembled to of pilin is called in the spirane structure of pili, and the molecular weight of this adventitia is about 10 ⁷Dalton.When the pili of purifying was dialysed with the phosphoric acid buffer of pH12, the irreversible pilin condensation product that resolves into of complete pili was called pilin oligomer (4).The size of the condensation product of these pili oligomer (molecular weight is about 600,000 dalton) is more much smaller than complete pili.

The single pilin that Diplococcus gonorrhoeae is expressed is isolated by Schoolnik and its co-worker at first, and order-checking (5).The gonococcus pilin is made up of following three zones: (a) the aminoterminal zone of high conservative (residue 1-53); (b) this district of region intermediate (residue 54-124) show limited amount sequence variations and (c) proteic third part be carboxyl terminal (residue 125-160), contain two sulphur rings of alterable height.

In the natural infection process, pili experiences and resembles and antigenic variation (3,6) when high-frequency.The genetics of this variation is complicated especially, has carried out extensive studies.Every strain gonococcus all has the ability to change the one-level aminoacid sequence of pili molecule, thereby changes the antigenic property of its pili.The molecule mechanism of being responsible for this variation relates to: the non-interchangeability reorganization (6) between expressing gene seat (pilE) and numerous (17 to 19) non-promotor silences (pilS) gene.Pili sequence (or its part) moves to an expressing gene seat from the pilS locus, produces new pili variation, thereby makes gonococcus can express extremely a large amount of pilins.

Opposite with gonococcus, meningococcus is only expressed two kinds of different types of pili, is called I class and II class pili.The same with gonococcus, meningococcus I class pili experience antigenicity and the time elephant variation (3).It is similar to gonococcus pili molecular weight (17-20kd) that I class pili shows, with reactive similar (epi-position of high conservative combines on this monoclonal antibody and the gonococcus pili) (3) of monoclonal antibody (SM1).Cloned many I class pili, the sequence of displaying acid sequence and gonococcus pili has high similarity (7).On the contrary, II class pili not with SM1 antibody response, and molecular weight also lower (13-16kd).Several bacterial strains of expressing II class pili show and can react with the polyclonal antiserum of anti-gonococcus pili.People such as Aho (7) have determined the sequence of II class pili recently.Trizonal first zone of Neisseria pili is identical basically.II class pilin has big disappearance to take place with the different hypervariable regions that are of I class pili and gonococcus pili.People such as Achtman (8) adopt the monoclonal antibody specific of I class or II class pili, have proved that some serogroups A isolates in Africa all combine with two kinds of antibody.Verified bacterial strain of expressing I class pili also amputation reticent I class pili gene (7).Combine and see, unicellular possibility of expressing two kinds of pilins simultaneously of meningococcus of these data promptings.

Contacting owing to thinking that pili has mediated with the initial of mucomembranous cell, is the disease of preventing the pili bacterium to cause as vaccine antigen these structures so be worth interested.In the crowd, carried out the test (9) of pili vaccine prevention traveler's diarrhea and gonorrhoea.Yet they are only effective to homologous strain so far.It is reported that some vaccines based on pili have been used for the disease that domestic animal suffers from, as IKB (blood-shot eye illness) (1), rot disease (1,10) and young pig (9) or calf (9) of poker suffered from diarrhoea.In the example of every kind of this class animal doctor research, the pili vaccine provides the protection of avoiding expressing homology rather than the attack of allos pili bacterial strain.

Gonococcus vaccine the earliest comprises whole microbe, seldom or not provides provide protection but provide.The exploitation of antigonococcique vaccine recently focuses on surface component, particularly pili (9,11) and the porin (P.I or Por) (11) of purifying.Yet so far, have only pili to demonstrate and can protect human body to be immune against attacks, and this be limited to the protection human body resist homologous strain (12).The denatured form (4) that has shown the gonococcus pili can produce in mouse and cavy can be at external antibody in conjunction with the allos pili.Yet, do not think that this is the method for viable commercial, because the gonococcus of the hair that carries disease germs in the liquid medium within (commercial production is essential) is difficult to growth (1).Based on the success of preliminary people's Attack Research, render a service the test (12) of having carried out gonococcus pili vaccine in the experiment an extensive placebo double blinding.In this experiment, this vaccine can not protect male volunteers to avoid gonococcal infection.Infer the most probable unhomogeneity (12) that is interpreted as pili of this situation.

Really the antigenic variation of gonococcus and meningococcal pilin is the major obstacle (3,13,14,15,16,17) to be enumerated repeatedly in the vaccine development of pili.Existing people proposes: the immunodominant epitope on the complete pili of assembling is two sulphur rings, and this ring presents maximum sequence variations (17,18).This has just explained the complete pili of the gonococcus bacterial strain Pgh3-2 that handles with formalin, in the reason (12) of Korea S's field experiment failure.In addition, some reference that this paper comprises are wherein referred to the pili of antivenom purification, or the segmental antiserum(antisera) of pilin, can be incorporated into sex change (Western trace) or isolating Neisseria hair, but not combine (16 with the allos pili of bacterium surface, 17,19,20).Also do not understand fully this whether be since in the pili of assembling the antigenic variation of epi-position or hidden due to.Some reports have been emphasized this point, and these report proofs are only in the external monoclonal antibody that presents functionally active, be only those not with allos pili bonded antibody (3).The complete pili of ox catarrhalis and plethora artiodactyl shape bacterium shows that it is possible (10) that pili causes protective immune response.Yet these vaccines can only protect the body opposing to express the attack (10,21) of the bacterium of homology pili rather than allos pili.Scientific circles are consistent to be thought and it seems vaccine based on pili, if possible, will only protect the body prevention to express the bacterium of homology pili.

The pili of recombinant expressed assembling is all described in many microbies, and it depends on suitable transhipment and assembled base because of (22).For Neisseria, do not find that in a continuous operon coding participates in the gene of pili assembling and translocator, so this method is infeasible.At European patent 202, the another kind of alternative method of selecting among the 260B1 is that (this host has had the required albumen of assembling different I V pili certain host bacterium; As Pseudomonas aeruginosa) the middle IV pili gene (23) of expressing.But report that as people such as Hoyne expressing and assemble the gonococcus pili is unsettled (24) on the Rhodopseudomonas adventitia.When growing in there is the liquid nutrient medium of selective antibiotic element in this kind recombinant bacterial strain, wild-type is carried disease germs the Rhodopseudomonas of hair can hypertrophy.The author is presented below:

" external source mePhe in the host strain (the N-methylbenzene L-Ala) consistency of pilin product will rely on the divergent degree of host and donor pilin line system.The unstable of the PAK/2PfS (Pseudomonas aeruginosa K/2PfS strain) of observed expression gonococcus pilin ... may show and express the limited method used in this example that is between mePhe pili kind." (24) owing to this result, expression gonococcus pili can not be thought the method for viable commercial in Rhodopseudomonas.

Elleman, Egerton and co-worker have described the vaccine with the complete pili exploitation opposing poker canker of plethora artiodactyl shape bacterium.The plethora artiodactyl shape bacterium infection of expression homology pili that the complete pili of field experiment demonstration has been protected zooprophylazis.This means that commercially available vaccine must contain 8 or 9 kind of different pili, to realize all-round protection.In order to make this method feasible, cloned the pili gene of plethora artiodactyl shape bacterium, and in expression in escherichia coli (25).Find that recombinant pilin combines with inner membrance.When test contains intestinal bacteria (supersound process is crossed) cell vaccine of express recombinant pilin in challenge trial, the antibody titers that recombinant Bacillus coli cells produces and similar (25) of the natural pili of purifying.Yet the agglutination titer that the recombinant Bacillus coli cells vaccine produces is obviously lower than (690 couples of .47,800) of complete pili, and is lower than the relevant titre (5,000-10,000) (25,26) of protectiveness.

After doing active the attack with plethora artiodactyl shape bacterium, with shown opposite of complete pili, the recombinant pilin vaccine can not demonstrate any significant protection activity.Emery and its co-worker show that the sex change of complete pili abolished pili is induced protection in animal ability (27).In addition, recovered this proteic effectiveness (causing the formation that protection avoids attacking the serious pathology in back) (28) with stain remover (octyl-β-D-glucoside) or the dissociated pili of low pH (2.2).And no matter in fact not significantly difference of antibody titers between each group.The author states " having one or more epi-positions relevant with quaternary structure (being destroyed with processed) ".They further state:

" e. coli expression product is done the vaccine failure, may be because its physical property occlusion on host cell membrane causes, though preliminary experiment shows that this is not to cause its invalid major cause (not having display data).The vaccine failure of another kind do to(for) e. coli expression product is interpreted as: the pilin unit may not can be incorporated into (suitably to present important epi-position) in the native conformation owing to the existence of leader sequence before the unit of expression.”(28)。

Elleman and its co-worker confirm to have the importance of conformational epitope on recombinant pilin, and confirm that they need increase immunne response by antigenic better presenting.They advise two kinds of methods: from this albumen of escherichia coli membrane purifying; Or in Pseudomonas aeruginosa, express this albumen, make in its fibril pili that can be assembled to cell surface.A kind of method in back is preferable, because " this will improve immunogenicity greatly and simplify proteic purifying " (25).

In addition, Elleman and its co-worker have also looked back employing pilin (protein subunit of pili) as the complete inferior candidate vaccine of the ripe fibril pili of plethora artiodactyl shape bacterium:

" ripe pili be it seems than the immune response that can cause stronger and suitable (that is K-aggegation) with the fibril pili protein subunit of dosage.About 5,000 K-agglutination titer is commonly considered as minimum and is replied on serology, and it is suitable that enough protective immunities that certain given plethora artiodactyl shape bacteria strain infects are replied and resisted to this minimum.The level of replying like this (reaching the higher order of magnitude) realizes by inoculating ripe pili vaccine easily, can not realize but inoculate isolating protein subunit (can only cause low-level serum K-agglutinating antibody) " (23).

People such as nearest Alves have reported additional data, the uncomfortable cooperation candidate vaccine (29) of prompting fibril pili protein subunit.When mouse immune has been inoculated the polynucleotide vaccine of coding intestinal bacteria CFA/I pili adhesin albumen (as pilin), induce the antibody of generation to induce the different of generation with natural complete CFA/I pili.In addition, the antibody of these anti-recombinant proteins is opposite with the antiserum(antisera) of anti-native protein, does not show any agglutination activity.

Except above-mentioned work, a kind of effective gonococcus or meningococcus vaccine have also been developed based on pili.Meningococcus vaccine is limited to those vaccines with serotype A, C, Y and W135 pod membrane.

Therefore, need evaluation to be used for protecting body to resist the component of the vaccine of the disease that causes by gonococcus or all serotypes of meningococcus.

Brief summary of the invention

Thus, an object of the present invention is to identify respectively that it may form the feasibility candidate vaccine of resisting these bacteriums by gonococcus and meningococcal derivatized suitable antigenicity structure.These candidate vaccines must be able to be induced, body identification and in conjunction with the antibody of the multiple isolate of each pathogenic Neisseria.

Following these and other objects of the present invention are by the clone and express gonococcus and meningococcal recombinant pilin (rpilin) is realized.

The invention still further relates to a kind of structure of plasmid, the I class pilin that this plasmid expression meningococcus is chimeric, wherein the aminoterminal zone of I parameningococcus pilin is by the corresponding aminoterminal regional replacement of gonococcus pilin.The amount of the meningococcus mosaic I class recombinant pilin of this plasmid expression is significantly higher than the amount of the I parameningococcus recombinant pilin of total length meningococcus pilE genetic expression.

In order to obtain the expression of the chimeric I class recombinant pilin of meningococcus, gomphosis DNA array at first is inserted in the suitable plasmid vector.Transform or appropriate host cell of transfection with this plasmid then.In one embodiment of this invention, host cell is a coli strain.Under the condition that makes the described chimeric I class recombinant pilin of host cell expression, cultivate this host cell then.

The invention still further relates to and make up a kind of plasmid, the II class pilin that this plasmid expression meningococcus is chimeric, wherein the carboxyl terminal zone of II parameningococcus pilin is by the corresponding carboxyl terminal regional replacement of gonococcus pilin.

In order to obtain the expression of the chimeric II class recombinant pilin of meningococcus, gomphosis DNA array at first is inserted in the suitable plasmid vector.Transform or appropriate host cell of transfection with this plasmid then.In one embodiment of this invention, host cell is a coli strain.Under the condition that makes the described chimeric II class recombinant pilin of host cell expression, cultivate this host cell then.

In another embodiment of the present invention, the recombinant pilin (gonococcus, meningococcus or chimeric) of separation and purifying is used to prepare vaccine composition, the recombinant expressed Nai Seshi that it comprises separation and purification belongs to pilin, and described pilin is made up of following three zones: (a) the aminoterminal zone of highly protecting; (b) region intermediate, this district shows limited amount sequence variations and (c) proteic third part carboxyl terminal, said composition can cause protective immune response in mammalian hosts.This vaccine composition can further comprise adjuvant, diluent or carrier.The example of adjuvant comprises aluminium hydroxide, aluminum phosphate, MPL ^TM, Srimulon ^TM, QS-21, IL-12 and Toxins,exo-, cholera.Give mammalian hosts with this vaccine composition with enough immunogenicity amounts, resist the disease that gonococcus or meningococcus are caused with the protection host.

Second aspect of the present invention comprises the method for preparing above-mentioned vaccine composition, comprise step: adding dosage is enough to make described vaccine composition to cause protective immune response in the human host, the recombinant expressed Nai Seshi of separation and purification belongs to pilin, and described pilin is made up of following three zones: (a) the aminoterminal zone of highly protecting; (b) region intermediate, this district shows limited amount sequence variations and (c) proteic third part carboxyl terminal.

The 3rd aspect of the present invention comprises the dna sequence dna of a kind of separation and purifying, the dna sequence dna that includes coding Diplococcus gonorrhoeae and the chimeric recombinant pilin of I class Neisseria meningitidis, described pilin has the aminoacid sequence of SEQ ID NO:2 amino acid/11-167 before processing, or has the aminoacid sequence of SEQ ID NO:2 amino acid 8-167 after being processed into maturation protein.

Aspect the 4th of the present invention, be included under the rigorous Southern hybridization conditions of height of standard, can with the dna sequence dna with purifying of separating of above-mentioned dna sequence dna hybridization.

The 5th aspect of the present invention comprises a kind of plasmid, the dna sequence dna that includes a kind of separation and purifying, this sequence is included under the rigorous Southern hybridization conditions of height of standard, can with the dna sequence dna of the dna sequence dna hybridization of the chimeric recombinant pilin of above-mentioned coding Diplococcus gonorrhoeae and I class Neisseria meningitidis, described pilin has the aminoacid sequence of SEQ ID NO:2 amino acid/11-167 before processing, or has the aminoacid sequence of SEQ ID NO:2 amino acid 8-167 after being processed into maturation protein.

The 6th aspect of the present invention comprises a kind of plasmid, and this plasmid contains the coding Diplococcus gonorrhoeae of above-mentioned separation and purifying and the dna sequence dna of the chimeric recombinant pilin of I class Neisseria meningitidis.

The 7th aspect of the present invention discloses with above-mentioned the 5th the described plasmid transformed host cells in aspect.

The 8th aspect of the present invention discloses a kind of method that produces the chimeric recombinant pilin of Diplococcus gonorrhoeae and I class Neisseria meningitidis, comprise: transform or transfection host cell with the described plasmid in above-mentioned the 5th aspect, and under the condition that allows the described chimeric recombinant pilin of host cell expression, cultivate this host cell.

The 9th aspect of the present invention comprises a kind of separation and the Diplococcus gonorrhoeae of purifying and the chimeric recombinant pilin of I class Neisseria meningitidis, before processing, have the aminoacid sequence of SEQ ID NO:2 amino acid/11-167, or after being processed into maturation protein, have the aminoacid sequence of SEQ ID NO:2 amino acid 8-167.

The of the present invention ten aspect discloses the purposes for preparing the medicine of immune Diplococcus gonorrhoeae with above-mentioned vaccine composition.

The 11 aspect of the present invention discloses the purposes for preparing the medicine of anti-Neisseria meningitidis with above-mentioned vaccine composition.

The 12 aspect of the present invention discloses the dna sequence dna of a kind of separation and purifying, the dna sequence dna that includes coding Diplococcus gonorrhoeae and the chimeric recombinant pilin of II class Neisseria meningitidis, described pilin has the aminoacid sequence of SEQ ID NO:4 amino acid/11-170 before processing, or has the aminoacid sequence of SEQ ID NO:4 amino acid 8-170 after being processed into maturation protein.

The 13 aspect of the present invention discloses the dna sequence dna of a kind of separation and purifying, under the rigorous Southern hybridization conditions of the height of standard, can hybridize with the described dna sequence dna in above-mentioned the 12 aspect.

The 14 aspect of the present invention comprises a kind of plasmid, and it comprises the separation of the 13 aspect of the present invention and the dna sequence dna of purifying.

The 15 aspect of the present invention comprises a kind of plasmid, comprises the dna sequence dna of the 12 aspect.

The 16 aspect of the present invention comprises the plasmid transformed host cells with the 14 aspect.

The 17 aspect of the present invention comprises a kind of method that produces Diplococcus gonorrhoeae and the chimeric recombinant pilin of II class Neisseria meningitidis, comprise: the plasmid with the 14 aspect transforms or transfection host cell, and cultivates this host cell under the condition that allows the described chimeric recombinant pilin of host cell expression.

The 18 aspect of the present invention comprises the Diplococcus gonorrhoeae and the chimeric recombinant pilin of II class Neisseria meningitidis of a kind of separation and purifying, before processing, have the aminoacid sequence of SEQ ID NO:4 amino acid/11-170, or after being processed into maturation protein, have the aminoacid sequence of SEQ ID NO:4 amino acid 8-170.

Brief Description Of Drawings

Fig. 1 has described the hair cell that carries disease germs of gonococcus (bacterial strain I756recA-) under the transmission electron microscope, after the anti-gonococcus recombinant pilin of cavy (obtaining) antiserum(antisera) (dilution in 1: 50 15 minutes) cultivation from bacterial strain pgh3-1, with (the dilution in 1: 5 of the anti-cavy IgG of donkey link coupled 12nm Radioactive colloidal gold, 30 minutes) reaction, dye with NanoVan again.Figure 1A is an anti-recombinant pilin cavy immune serum (the 6th week); Figure 1B is normal guinea pig serum (the 0th week); Fig. 1 C does not have first antibody (primary antibody).

Fig. 2 has described the anti-gonococcus recombinant pilin of cavy (obtaining from bacterial strain Pgh3-1) antiserum(antisera), the gonococcus cell (bacterial strain I756recA-) of the hair that carries disease germs is adhered to the effect of human cervical cancer 1 cell (ME180 clone).Fig. 2 A has described the anti-gonococcus recombinant pilin of cavy antiserum(antisera) to this inhibition of adhering to (the 6th week); Fig. 2 B has described normal guinea pig antiserum(antisera) can not prevent from the to carry disease germs gonococcus cell attachment cervical cell (the 0th week) of hair.Irised out the size that the representativeness that is incorporated into the bacterium of cervical cell in every group of experiment is gathered together.Every group of experiment shown following four the different visuals field of same experiment condition.Every group of experimental guinea pig antiserum(antisera) done 1: 10,000 dilution.

Fig. 3 has described the hair cell that carries disease germs of meningococcus (bacterial strain H355) under the transmission electron microscope, after I class recombinant pilin (obtaining from the bacterial strain H44/76) antiserum(antisera) chimeric with the cavy meningococcemia (dilution in 1: 60 30 minutes) cultivated, be coupled to (the dilution in 1: 5 of 12nm Radioactive colloidal gold with the anti-cavy IgG of donkey, 30 minutes) reaction, dye with NanoVan again.Fig. 3 A is an anti-recombinant pilin cavy immune serum (the 6th week); Fig. 3 B is normal guinea pig serum (the 0th week); Fig. 3 C does not have first antibody.Cell and antiserum(antisera) are fixing before cultivating.

Detailed Description Of The Invention

The present invention relates to contain the vaccine combination of gonococcus or meningococcus recombinant pilin. Although the above has discussed many, still determine the purposes of this recombinant pilin of research expression in escherichia coli. Surprisingly, these recombinant pilins demonstrate the feature of candidate vaccine.

First report that is described in clone gonococcus pilE gene in the Escherichia coli is nineteen eighty-two (30). From then on, the characterization of molecules of pilE has been carried out a large amount of following genic laboratory researches, these genes are being controlled the expression of pilin, the transhipment of pilin, the variation of pilin sequence and the host of pili and are being sticked together characteristic. Yet the neither one report has been described the immune response of purifying or the recombinant expressed pilin of recombinant pilin.

Following embodiment 2 has described pilE gene cloning and the expression of coding gonococcus recombinant pilin. Expression is by realizing with the coli strain of the Plasmid Transformation called after TOP10F ' that contains the pilE gene. After successful clone and the expression, by the order-checking confirmation of pilE gene and the homogeny of native sequences. In order to help the clone, introduced NcoI2 site (requiring to modify a base). As a result, the second amino acid in the long signal peptide of seven amino acid becomes aspartic acid from asparagine.

The plasmid (called after pPX2000) that contains the pilE gene among the embodiment 2 includes an amicillin resistance (Amp^R) mark. As described in example 3 above, make up another and contained kalamycin resistance (Kan^R) mark, rather than Amp^RPlasmid. This plasmid called after pPX2002 after it transforms Escherichia coli TOP10F ' bacterial strain, has expressed the gonococcus recombinant pilin, and level is similar to pPX2000 and (contains Amp^RMark) resulting level.

As described in Example 4, with similar scheme constructs called after pPX2003 contain a plasmid of meningococcus I class fimbriae gene. Introduce NcoI site (CC ATG G), cross over the initiating terminal of code signal peptide gene. This becomes aspartic acid (the first residue still is methionine) with signal peptide the second amino acid residue from asparagine. Also comprise an Amp^RMark. Behind this construction conversion Escherichia coli TOP10F ' bacterial strain, expressed meningococcus I class recombinant pilin. Yet the level of its expression significantly is lower than the gonococcus recombinant pilin that pPX2000 or pPX2002 obtain. Not bound by theory, this lower expression may be because due to a large amount of inverted repeats (coming across among the restructuring I class pilE).

In order to increase the expression of meningococcus pilin, as described in Example 5, made up a chimeric plasmid. Front 60 amino acid of coding meningococcus I class recombinant pilin are replaced with the respective regions of gonococcus DNA among the pPX2002 among the pPX2003. Resultant Amp^RPlasmid called after pPX2004, its nucleotide sequence that has is seen SEQ ID NO:1. E. coli k12 strain with pPX2004 Plasmid Transformation called after TOP10F '. After inducing, the amount of the meningococcus recombinant pilin of expressing with pPX2003 is compared, and the chimeric recombinant pilin of expression has had remarkable increase. The amount of the gonococcus recombinant pilin that the expression of this chimeric construct thing and pPX2002 express is suitable. Chimeric I class recombinant pilin 167 amino acid of length (comprising burst) (SEQ ID NO:2) conform to the size of estimating.

The e. coli k12 strain of called after TOP10F ' (containing recombinant plasmid pPX2004), be deposited in by American type culture collection by the applicant on January 27th, 1998,10801 University Boulevard, Manassas, Virginia 20110-2209, U.S.A., and specifying the ATCC accession number is ATCC98637.

As described in Example 6, the chimeric plasmid of structure, 3 ' end of its meningococcus II class pilE gene is replaced by gonococcal respective regions. Specifically, the DNA of coding two sulphur rings (last 22 amino acid of the meningococcus II class pilin of meningococcus bacterial strain FAM18) is added that by similar (but larger) zone of gonococcus (pilE) DNA of gonococcus bacterial strain Pgh3-1 among the pPX2000 extention (amount to 44 amino acid) in c-terminus zone replaces among the pPX8001. The Amp that the result obtains^RPlasmid called after pPX8017, the nucleotide sequence that it has see SEQ ID NO:3, and wherein nucleotides 1-378 is from meningococcus II class, and nucleotides 379-510 is from gonococcus. Transform the e. coli k12 bacterial strain of name TOP10F ' with plasmid pPX8017. After inducing, expressed chimeric II class recombinant pilin, its length is 170 amino acid (burst that comprises 7 amino acid longs) (SEQ ID NO:4), and wherein amino acid/11-126 is from meningococcus II class, and amino acid/11 27-170 is from gonococcus. This chimeric II class recombinant pilin conforms to the size of expectation. Consider for the clone, a NcoI site is introduced, so second amino acid in the burst is become glutamic acid from lysine. Estimate that this variation can not have any impact to antigenicity or immunogenicity.

Carry the sample of the e. coli k12 strain (being called TOP10F ') of recombinant plasmid pPX8017, be preserved in American type culture collection (ATCC) (10801 University Boulevard on April 15th, 1999 by the applicant, Manassas, Virginia 20110-2209, U.S.A.), its ATCC accession number is ATCC207199.

Host cell-carrier system of describing in detail in embodiment 2-6, various host cell-carrier system all is suitable for expressing gonococcus used in the vaccine of the present invention, meningococcus and chimeric recombinant pilin. Carrier system should be compatible with used host cell. The host cell that is fit to comprises: with DNA, cosmid DNA or the bacterium of having a liking for the thallus DNA conversion; Virus is such as vaccinia virus and adenovirus; Yeast such as Pichia cell; Insect cell such as Sf9 or Sf21 cell; Or mammal cell line such as Chinese hamster ovary cell; And other conventional organism.

Various routine is transcribed and is translated element and can be used for the host cell carrier system. PilE DNA is inserted an expression system, and promoter and other control element are connected to the specific site of this carrier, when this plasmid vector was inserted into host cell, host cell can be expressed pilE DNA like this.

According to used host cell carrier system, can or infect plasmid is introduced host cell by conversion, transduction, transfection. Then, under can expressing the condition of recombinant pilin, host cell cultivates host cell.

The invention still further relates to the dna sequence dna of a kind of separation and purifying, the I class recombinant pilin that the dna sequence encoding meningococcus that it comprises is chimeric, the aminoterminal zone of this albumen from gonococcus pilE gene and its central authorities and the c-terminus zone from meningococcus pilE gene (SEQ ID NO:1). The chimeric I class recombinant pilin of meningococcus before the nucleotides 1-501 coding processing among the SEQ ID NO:1; Become the chimeric I class recombinant pilin of meningococcus of maturation protein after nucleotides 22-501 coding processing. The invention still further relates to the chimeric I class recombinant pilin of meningococcus, this albumen has the amino acid sequence (becoming maturation protein after the processing) of amino acid 8-167 among the amino acid sequence (processing before) of amino acid/11-167 among the SEQ ID NO:2 or the SEQ ID NO:2. About 10% lacks burst in the total protein that is produced by the chimeric I class recombinant pilin construction of gonococcus recombinant pilin or meningococcus, and it is removed by processing.

The invention still further relates to the dna sequence dna of a kind of separation and purifying, the II class recombinant pilin that the dna sequence encoding meningococcus that it comprises is chimeric, the c-terminus zone of this albumen from gonococcus pilE gene and its central authorities and the aminoterminal zone from meningococcus pilE gene (SEQ ID NO:3). The II class recombinant pilin (before the processing) that nucleotides 1-510 coding meningococcus among the SEQ ID NO:3 is chimeric; The chimeric II class recombinant pilin (becoming maturation protein after the processing) of nucleotides 22-510 coding meningococcus. The present invention also relates to the chimeric II class recombinant pilin of meningococcus in addition, and this albumen has the amino acid sequence (becoming maturation protein after the processing) of amino acid 8-170 among the amino acid sequence (processing before) of amino acid/11-170 among the SEQ ID NO:4 or the SEQ ID NO:4.

The gomphosis DNA array in being included in pPX2004 and pPX8017 (the II class recombinant pilin that the I class recombinant pilin that the meningococcus of encoding respectively is chimeric and meningococcus are chimeric), the present invention comprises that also (because genetic code Feng Yuxing) biologically is being equivalent to the DNA sequence of the chimeric recombinant pilin of coding, that is to say, the feature of these other dna sequence dna, although it is different from nucleotide sequence shown in this article that it is nucleotide sequence, coded identical of the amino acid sequence of the protein that they are coded and the dna sequence dna among SEQ ID NO:1 or the SEQ ID NO:2.

Specifically, the sequence that the present invention includes those and SEQ ID NO:1 or SEQ ID NO:3 has enough homogenies, thereby can be at the lower dna sequence dna that can hybridize of highly rigorous Southern hybridization conditions (condition described in the people such as Sambrook (31)).

The present invention also comprises such dna sequence dna: the amino acid sequence of its coding is different from the recombinant pilin of the chimeric I class of meningococcus or II class, but biologically is being equivalent to the amino acid sequence (SEQ ID NO:2 or SEQ ID NO:4) of one of these albumen. These amino acid sequences can be described as with those sequences of chimeric recombinant pilin of equal value biologically, if the difference of their sequences is the small disappearance, insertion of recombinant pilin sequence or substitutes, thereby being compared with three grades of conformations of recombinant pilin, three grades of conformations of this sequence there is no variation.

For example, the residue (such as glycine) that another hydrophobicity of codon available code of amino acid alanine (a kind of hydrophobic amino acid) is relatively poor, or the codon of the stronger residue (such as valine, leucine or isoleucine) of hydrophobicity substitutes. Similarly, change can be to cause a kind of electronegative residue alternative another (substituting glutamic acid such as aspartic acid) or a kind of positively charged residue to substitute another (such as lysine place of arginine) and with substituting between the approximate residue of hydrophily, also can expect to produce biologically suitable product. The nucleotides that expectation causes protein molecular N-end or C-end to change changes, but can not change the activity of protein.

In addition, the variation in known Variable Area is biologically of equal value, and wherein three of conserved region grades of conformations there is no change (with comparing of those recombinant pilins). Biologically the another kind of equivalent sequence is defined as: the sequence of the immune response of the generation cross reaction of still having the ability. Specifically, the I class that meningococcus is chimeric and II recombinant pilin can lengthen by the corresponding insert with the gonococcus pilin or shorten and modify, as long as the chimeric recombinant pilin of modifying still has the ability that produces the cross reaction immune response.

The modification of each suggestion is to adopt the ordinary skill in the art, whether has kept structure and biologically active as measuring coded product. So, used term " the I class recombinant pilin that meningococcus is chimeric " or " the II class recombinant pilin that meningococcus is chimeric " in specification or claims is appreciated that this term has comprised and causes producing biologically all these modifications and the variation of albumen of equal value.

As described in Example 7, the gonococcus recombinant pilin is combined with the colibacillary cell membrane of expressing this albumen. Multiple detergent can the colibacillary recombinant pilin of selective dissolution, comprises Empigen^TM BB、 Triton ^TMThe Triton of X-100, reduction^TMX-100, octyl-β-D-glucopyranoside (OG), Zwittergent^TM3-10 or 3-14. After centrifugal, the dialysis and separate with the post classification, obtain the recombinant pilin of purifying.

As described in Example 8, chimeric I class recombinant pilin separates and purifying by the Bacillus coli cells that breaks, and by centrifugal clarification, filters, and separates at two column fractionations.

As described in Example 9, the chimeric II class recombinant pilin of meningococcus can separate and purifying by the Bacillus coli cells that breaks, by centrifugal clarification, and dialysis, and two column fractionations separation.

As described in Example 10, the gonococcus recombinant pilin of purifying is carried out the N-end order-checking of repetition. The consensus amino acid sequence that the result that the order-checking of 20-40 aminoterminal residues provides and dna sequence dna are derived. The molecular weight that mass spectrography records recombinant pilin (burst is arranged) is 18,006 dalton, meets very much quality 17,981 dalton (estimating according to aminoacid ingredient) of expectation. On the contrary, when recombinant pilin carried out the size exclusion column chromatography with detergent, the apparent molecular weight that obtains was 68,899 dalton. This aggegation of prompting recombinant pilin. With the PBS recombinant pilin of dialysing, remove detergent, the apparent molecular weight of gained material is 452,349 dalton (measuring with gel filtration) as a result. This points out it to experience further aggegation.

As described in embodiment 11, with the gonococcus recombinant pilin immune guinea pig or the mouse acquisition immune serum of purifying.As listing among the embodiment 12, the Western engram analysis shows that the antiserum(antisera) of anti-recombinant pilin is incorporated into the full cell lysate of the gonococcus cell system of the hair that carries disease germs, and does not combine with the cell lysate of no pili.On the contrary, the antiserum(antisera) of antibiotic hairless protein oligomer be incorporated into the hair that carries disease germs with the cell lysate of no pili.

Describe in detail as embodiment 13, when using elisa assay, the antiserum(antisera) of the anti-gonococcus recombinant pilin of this blended has very high terminal point titre to the gonococcus recombinant pilin that is incorporated into purifying.Embodiment 13 also describes the effect of various adjuvants in detail.As recombinant pilin list MPL ^TM, MPL ^TMWith aluminum phosphate or Stimulon ^TMWhen QS-21 makes adjuvant, in mouse, obtain good humoral immunoresponse(HI).

As listing among the embodiment 14, full cell ELISA shows that the antiserum(antisera) of anti-recombinant pilin is incorporated into the fimbriate cell of specific gonococcus bacterial strain, but debond does not have the cell of pili in homogenic specific gonococcus bacterial strain.

As described in embodiment 15, mouse is carried out immunization in the nose with gonococcus recombinant pilin (have or do not have natural Toxins,exo-, cholera).Mouse is with after the recombinant pilin immunization of no adjuvant, and the antigen ELISA of the pooled serum of generation has recorded significant immunne response; This replying owing to adding natural Toxins,exo-, cholera increases.Pooled serum is to very low in conjunction with complete fimbriate gonococcus cell ELISA titre; When mouse during with natural Toxins,exo-, cholera immunization, this combination improves greatly.

As described in embodiment 16, immuno-electron microscope shows pili fibril length (direction) combination of the antibody of anti-recombinant pilin along the gonococcus surface.This prompting internal antibody is incorporated into the epi-position that bacterium surface exists.

Embodiment 17 shows, compares with the result of reorganization pili oligomer antiserum(antisera) gained, and is higher with the allos capillary bacterium isolate bonded recombinant pilin antiserum(antisera) titre of carrying disease germs.With pH12 phosphoric acid buffer dialysis recombinant pilin, make recombinant pilin be transformed into the recombinant pilin oligomer.

Pili has mediated gonococcus and has combined with the preliminary of people's mucomembranous cell.So, if having the ability to cause, a kind of antigen stop gonococcus to be adsorbed in the antibody of people's mucomembranous cell, just proved that this antigen is candidate vaccine.

As described in example 18 above, the anti-recombinant pilin antiserum(antisera) of the cavy gonococcus that suppressed the expressing heterologous pili significantly combines with human cervical cancer 1 is epithelial.So the generation of gonococcus pili and the infectivity relevant (2,3,32) of this bacterium.

These data show; recombinant pilin can produce the antibody that is incorporated into various pili on the complete gonococcus cell; and this antiserum(antisera) shows functionally active (inhibition bacterial adhesion), and this will protect the people of immunization to resist gonococcus and settle down and infect (32,33).The front reported, was that immunogenic (23,25,28) are arranged with the Bacillus coli cells immunization of the recombinant pilin of expressing plethora artiodactyl shape bacterium, but can not protects the body opposing to attack.Because these results, these investigators no longer adopt the recombinant pilin subunit, and agree with adopting the pili of assembling.Yet the Notes of Key Data described herein has induced a kind of human body of protecting to prevent the immunne response that gonococcus is settled down behind the recombinant expressed pilin purifying.So these data have supported that recombinant pilin is the viewpoint that is used to resist gonococcal candidate vaccine.

As described in embodiment 19, to the N end order-checking of the chimeric I class of meningococcus recombinant pilin.The result that the order-checking of these 35 aminoterminal residues provides is consistent with the dna sequence dna deduced amino acid.The molecular weight of chimeric recombinant pilin (signal peptide is arranged) is 17,659 dalton by mass spectroscopy, with suitable according to 17,676 dalton of aminoacid component prediction.On the contrary, when the chimeric I class of meningococcus recombinant pilin carried out the size exclusion chromatography analysis with stain remover, obtaining apparent molecular weight was 69,480 dalton.Compare with the gonococcus recombinant pilin, the aggegation of the chimeric I class of this prompting meningococcus recombinant pilin.

Describe in detail as embodiment 20, when use elisa assay, the blended antiserum(antisera) of the chimeric I class of meningococcemia recombinant pilin all has very high terminal point titre to meningococcus I class recombinant pilin and the meningococcus cell that carries disease germs mao.As detailed described among the embodiment 20, adjuvant, particularly Stimulon ^TMQS-21 is incorporated into the chimeric I class of meningococcus recombinant pilin and the meningococcus cell of the hair that carries disease germs to the antiserum(antisera) of the chimeric I class of meningococcemia recombinant pilin, has produced significantly and has replied.

As described in embodiment 21, immuno-electron microscope shows length (direction) combination of the antibody of the chimeric I class of meningococcemia recombinant pilin along the meningococcus pili.This prompting internal antibody is incorporated into the epi-position that bacterium surface exists.

In embodiment 4, show the antiserum(antisera) identification of anti-gonococcus recombinant pilin and be incorporated into the meningococcus cell of the hair that carries disease germs.Show that in embodiment 22 antiserum(antisera) of the chimeric I class of the meningococcemia of generation recombinant pilin can be incorporated into the gonococcus cell of the hair that carries disease germs.

As described in embodiment 23,, can help prevention meningococcemia in the body with the chimeric I class of cavy meningococcemia recombinant pilin antiserum(antisera) passive immunization children mouse.The level of settling down of bacterium significantly reduces in the animal of accepting this immune serum.In addition, the immunne response that produces with recombinant expressed pilin can be protected the meningococcus of opposing expressing heterologous pilin in vivo.

As described in embodiment 24, with having or not with immunized mice in the chimeric I class of the meningococcus of the Toxins,exo-, cholera recombinant pilin nose, Toxins,exo-, cholera wherein be mutant (L-glutamic acid of its amino acid sites 29 is replaced with Histidine) (CT-CRM, E29H).After the recombinant pilin inoculation of mouse with no adjuvant, the pooled serum of generation has recorded tangible immunne response in antigen ELISA; This is replied by adding mutant CT-CRM, E29H Toxins,exo-, cholera and improving.

As described in embodiment 25, the subcutaneous immunization of mouse contains MPL ^TMShow behind the chimeric I class of the meningococcus of the adjuvant recombinant pilin: suppressed Neisseria meningitidis I class bacterial strain settling down at the mouse nasopharynx.

As described in embodiment 26, the Western engram analysis shows the antiserum(antisera) that obtains from the cavy with the chimeric II class of meningococcus recombinant pilin immunization, is incorporated into the full cell lysate of the meningococcus cell (expressing I class or II class pilin) of the hair that carries disease germs.

As described in embodiment 27, inductive is incorporated into the meningococcus cell of homology bacterial isolates at the antiserum(antisera) of the chimeric II class of partially purified meningococcus recombinant pilin.

In sum, these data have been supported recombinant pilin, and particularly chimeric I class of meningococcus and II class recombinant pilin are the viewpoints of the candidate vaccine of anti-Neisseria meningitidis.

The gonococcus recombinant pilin can be used for preparing the vaccine of the disease that protection Mammals opposing gonococcus causes.The chimeric recombinant pilin of meningococcus, meningococcus chimeric I class recombinant pilin and the chimeric II class of meningococcus recombinant pilin can be used for preparing the vaccine of the disease that protection Mammals opposing Neisseria meningitidis causes.

In addition, different Neisseria gonorrhoeae bacterial classifications are resisted in cross protection, can be by carrying out immunization with the vaccine that contains gonococcus recombinant pilin (disease that the opposing of protection Mammals is caused by Neisseria meningitidis); Or with containing the meningococcus recombinant pilin, the vaccine of meningococcus chimeric I class recombinant pilin or the chimeric II class of meningococcus recombinant pilin carries out the disease that the opposing of immunization protection Mammals is caused by gonococcus.

These vaccine compositions comprise the recombinant pilin of a kind of separation and purifying, and this vaccine composition can cause protective immune response in mammalian hosts.

The vaccine that contains recombinant pilin can be mixed with the carrier of acceptable diluent on the immunology or conventionally form, prepares injectable solution or suspension.The antibody horizontal that this vaccine causes can improve by adopting some adjuvant, as Stimulon ^TMQS-21 (Aquila Biopharmaceuticals, Inc., Framingham, MA), MPL ^TM(3-O-deacylated tRNA monophosphoryl lipid A; RIBI ImmunoChem Research, Inc., Hamiltion, MT), aluminum phosphate, aluminium hydroxide, IL-12 (Genetics Institute, Cambridge, MA) and Toxins,exo-, cholera (wild-type or mutant form, as the L-glutamic acid in amino acid 29 sites by another amino acid, preferably Histidine displacement, U.S. Provisional Patent Application 60/102,430).

Vaccine of the present invention can be by the injection of traditional form, goes into human body as subcutaneous, intraperitoneal or intramuscularly, and in the oral cavity, mucous membrane, nose or vagina give, to induce the initiative immunne response of the disease that protection body opposing gonococcus or meningococcus cause.The dosage that gives is determined by method known to those skilled in the art.By the vaccine of single dose, maybe need to give several times booster dose and give protection.

In order to make the present invention's easy to understand more, list following examples.These embodiment only are for purposes of illustration, do not constitute any restriction to the scope of the invention.

Embodiment

Press the described schemes of people such as Sambrook, used standard molecular biological technique (31).

Embodiment 1

Bacterium and cell cultures

Bacterium and culture condition

The gonococcus isolate derives from Tampa, FL; Ottawa, Canada; Washington, D.C.; Seattle, WA; Rochester, NY; Chapel Hill, NC; And Evanston, IL.The meningococcus isolate derives from Chapel Hill, NC; And Bilthoven, Netherlands.

Bacterium preserve with lyophilized form or-70 ℃ freezing up to use.When growing on solid medium, agar plate is at wet environment and 5% (v/v) CO ₂37 ℃ of incubators in cultivate.Gonococcus and Neisseria meningitidis are at GC substratum (Difco Laboratories, Detroit, MI) go up growth, this substratum hemoglobin-free, but be added with glucose (400mg/L), glutamine (10mg/L), cocarboxylase (20 μ g/L) and iron nitrate (500 μ g/L).Meningococcal fluid suspension culture thing is grown in 37 ℃ of shaking culture casees (70RPM) in lacking the same substratum of agar.In culture of isolated in the experiment of the meningococcus of mouse nose tissue homogenate, bacterium grows on above-mentioned GC substratum, and contains following blended antibiotic (Difco): colistin sulfate (75 μ g/ml), nystatin (125 μ g/ml), vancomycin (30 μ g/ml) and lactic acid Trimethoprim BP (125 μ g/ml).By the gonococcus that colonial morphology is identified the hair that carries disease germs, single bacterium colony goes down to posterity to keep phenotype every day.Meningococcus cell pili generation state is by (NY) sample of (pH830 minute) carries out the transmission electron microscope assessment for Nanoprobes, Stony Brook to NanoVan dyeing.Intestinal bacteria are grown on SOB agar, SOB agar is made up of following: 20g/L Bacto Tryptones (Difco), 5g/L yeast extract (Difco), 0.6g/L NaCl, 0.2g/L KCl and 1% (w/v) agar (pH7.5), or growth in SOB meat soup (not adding agar).In some experiments, the Bacto Tryptones Hysoy of same amount ^TM(Sheffield Products, Norwich NY) replace.

Epithelial cell is cultivated

(ATCC, Beltsville MD) are a kind of epidermoid carcinoma to ME-180 clone, originally are derived from cervical cancer.Make this cell add 10% (v/v) foetal calf serum (Sigma, St.Louis, MO), penicillin G (RPMI1640 (the Gibco BRL of (Gibco BRL), L-Streptomycin sulphate (1mg/mL) (Gibco BRL) and 2mM L-glutaminate of 1000 units/mL), Gaithersburg, MD) in, 37 ℃ of 5% (v/v) CO ₂Down growth of wet environment.Division in the every 3-4 of cell days once.

Embodiment 2

Clone and the expression of gonococcus in intestinal bacteria

With the freezing sample of gonococcus bacterial strain Pgh-1 of the hair that carries disease germs source as pilE DNA in the PCR reaction.The primer of the complete pilin of following identification (comprising leader sequence) 3 ' and 5 ' end: 5 ' CCC CGC GCC ATG GAT ACC CTT CAA AAA GGC 3 ' (PILEFWD) (SEQ IDNO:5) and 5 ' GGG CCT GGA TCC GTG GGA AAT CAC TTA CCG 3 ' (PILEREV) (SEQ ID NO:6) used in the amplification of pilE gene.The PCR product that obtains contains a NcoI site at the section start of pilE coding region, contains a BamHI site endways.Consider the clone, this gene is introduced in the NcoI site.Second amino acid whose change that this will cause signal sequence becomes aspartic acid (GAT) from l-asparagine (AAT).Because amino acid 2 is parts of signal peptide, this signal peptide is rived in the normal process of maturation protein, so this change expection does not have any influence to antigenicity or immunogenicity.The PCR product cloning is arrived pCR ^TMThe IITA cloning vector (Invitrogen, Carlsbad, CA) in, connect, be transformed into (Invitrogen) among the intestinal bacteria TOP10F '.Screen bacterium colony containing the dull and stereotyped of 100 μ g/mL penbritins or contain on the flat board of 50 μ g/mL kantlex.From the overnight culture of these transformant, isolate plasmid DNA, and analyze with enzyme EcoRI and NotI restrictive diges-tion.

Contain the segmental clone of correct size insertion to four and make dna sequence analysis, prove conclusively the existence of pilE PCR dna fragmentation.Clone #17, called after pPX1999 is as the source of pilE gene.The plasmid DNA of pPX1999 and pTrcHisA (Invitrogen) is with NcoI and BamHI digestion with restriction enzyme, with this dna fragmentation gel separation, connects and is transformed among the intestinal bacteria TOP 10F '.Screen the amicillin resistance bacterium colony, separate the plasmid DNA of new transformant, carry out the DNA restriction analysis with BamHI and NcoI restriction enzyme.Two clones with correct unrestricted model are made dna sequence analysis.These two clones have correct dna sequence dna, called after pPX2000.

Shake in bottle or the fermentor tank at the SOB that is added with 100 μ g/mL penbritins and 12 μ g/mL tsiklomitsins and grow in order to test the expression of recombinant pilin, make the culture that contains these clones.When shaking bottle, intestinal bacteria grow to A in the 1L substratum ₆₀₀=0.9-1.0.The isopropyl-(IPTG) that adds final concentration and be 1mM is induced the expression of recombinant pilin, making growth continue 1-4 hour, this moment centrifugal (13,689 * g, 20 minutes, 4 ℃) collecting cell ,-20 ℃ of preservations.For fermentor tank, the overnight culture of a flat board is inoculated into contains the shaking in the bottle of 500mL substratum, continue overnight incubation.Then this liquid culture is inoculated into the Biostat B fermentor tank that contains the 8.9L substratum (Braun Biotech, Allentown, PA) in.When containing HySoy ^TMSubstratum when having added the glucose of final concentration 1% (w/v), the growth of bacterium is improved in the fermentor tank.When culture reaches A ₆₀₀=1.0 o'clock, adding final concentration was the IPTG of 1mM, makes continued growth 1-4 hour, centrifugal then (13,689 * g, 20 minutes, 4 ℃) harvested cell.Abandon nutrient solution ,-20 ℃ of cell precipitations are preserved.After inducing with IPTG, significantly increase the expression of recombinant pilin, induced the back to reach highest level in 3-4 hour.

The sample of inducing culture thing carries out the polyacrylamide gel electrophoresis analysis with sodium laurylsulfonate (SDS-PAGE).Recombinant pilin is observed with Coomassie blue stain, and (Emeryville CA) carries out the checking of Western trace and identifies it for clone #A33020023, Biospacific with gonococcus pili monoclonal antibody specific.

Embodiment 3

Structure contains the gonococcus reorganization pilE plasmid of kalamycin resistance mark

Make up a plasmid, its Amp ^RMark Kan ^RToken-replacement.Except that in particular the following, with the flow process among the embodiment 2.The pTrcHisA plasmid DNA is carried out the PCR reaction, adopt the TrcFXba primer, 5 ' GGC TCT AGACTG TCA GAC CAA GTT TAC TC3 ' (SEQ ID NO:7) and TrcRXba primer, 5 ' GGC TCT AGATTG AAG CAT TTA TCA GGG3 ' (SEQ ID NO:8).The sequence encoding XbaI restriction site of band underscore.About 3.5kb PCR product contains pTrcHisA DNA and deducts the penbritin coding region.(New England Biolabs, Beverly MA) have carried out another PCR reaction, adopt the KanFXba primer, 5 ' GGC to the pACYC177 plasmid DNA TCT AGATAA ACAGTA ATA CAA GGG G3 ' (SEQ ID NO:9) and KanRXba primer, 5 ' GGC TCT AGATTA GAA AAA CTC ATC GAG C3 ' (SEQ ID NO:10).Equally, the sequence encoding XbaI restriction site of band underscore.The PCR product of about 860bp contains kalamycin resistance gene.Two kinds of PCR products purifying from the sepharose obtains, and with two kinds of DNA of XbaI digestion with restriction enzyme, extracts and couples together.A ligation thing is transformed among the intestinal bacteria TOP10F ', and a transformation mixture inoculation contains the SOB flat board of 30 μ g/mL kantlex.

To amount to 48 Ka ^RBacterium colony is containing streak culture (in duplicate) on the SOB flat board of penbritin or kantlex.As expected, all Kan ^RBacterium colony all is amp-S.Because clone's design is symmetric, separates the kantlex inset that has obtained two kinds of orientations.Filter out and original identical clockwise kantlex inset of penbritin gene, be used for further research, be called pZ564.To contain the DNA zone of lacIq gene, trc promotor and multiple clone site among the pZ564, with similar area (also the containing pilE) displacement of pPX2000 plasmid, method is as follows: with SphI and XmnI digestion with restriction enzyme pZ564 and pPX2000.About 2.2kb dna fragmentation of gel-purified pPX2000 and about 2.6kb dna fragmentation of pZ564 couple together, and are transformed among the intestinal bacteria TOP10F '.The correct plasmid that obtains is called pPX2002.

When selecting microbiotic when penbritin changes kantlex into, can see similar time course of inductive and recombinant pilin expression level.

Embodiment 4

Clone and expression meningococcus I class pilE in intestinal bacteria

Because the high homology (7) of gonococcus and meningococcus I class pilin DNA and aminoacid sequence, assessed the ability that gonococcus recombinant pilin antiserum(antisera) is incorporated into the pili of meningococcus cell expressing with full cell ELISA.It is 151,100 that this antiserum(antisera) shows the carry disease germs titre of hair cell of the Neisseria meningitidis that is incorporated into bacterial strain H355.

This combination is seemingly at pilin, because the Western trace of this strain whole-cell lysate is shown, has only the single band that moves altogether with pilin can be in conjunction with this antiserum(antisera) (not having display data).Other meningococcus bacterial strain shows low titre in full cell ELISA in a large number, but transmission electron microscope does not prove the existence of pili.According to these data, the I class pilin of decision clone and recombinant expressed Neisseria meningitidis.Originally, adopted the identical strategy of gonococcal pilE.Except that in particular the following, use the flow process among the embodiment 2.

From Neisseria meningitidis bacterial strain H44/76 genomic dna amplification I class pilE, adopt following primer: 5 ' CCC CGC GCC ATG GAC ACC CTT CAA AAA GGT TTT ACC3 ' is (SEQ ID NO:11) and 5 ' GGG CCT GGA TCC GAG TGG CCG TGG AAA ATCACT TAC CGC3 ' (NMRPILE) (SEQ ID NO:12) (NMFPILE).As estimating, obtain the PCR product of about 600bp DNA.A PCR reaction product is inserted into pTrcHisA with BamHI and NcoI digestion with restriction enzyme.DNA electrophoresis on sepharose of digestion, the gel-purified dna fragmentation.Then dna fragmentation is coupled together, be transformed among the intestinal bacteria TOP10F '.With BamHI and NcoI restriction enzyme the amicillin resistance clone being carried out the miniplasmids preparation analyzes.The clone who expresses correct restrictive diges-tion pattern is called RZ1142, and plasmid is called pPX2003.

After inducing, when analyzing full cell lysate, observe molecular weight and be about 15,000 daltonian Coomassie blue stain polypeptide with SDS-PAGE.Full cell lysate with four kinds of clones of SDS-PAGE and these clones of Western engram analysis shows, has a kind of albumen, it have suitable mobility and with the responding property of polyclonal antiserum of the complete pili of anti-gonococcus bacterial strain LB2.The I class recombinant pilin length of purifying is 167 amino acid.Yet the expression level of this recombinant pilin is starkly lower than the pPX2000 of growth under the same terms or the level that pPX2003 obtains.The dna sequence dna of analyzing among the reorganization I class pilE shows a large amount of inverted repeats are arranged, the reason of this albumen low expression level in these possible explanation intestinal bacteria.

Embodiment 5

In intestinal bacteria, make up and express gonococcus and the chimeric pilE of meningococcus I class

In order to increase the expression of meningococcus pilin, with preceding 60 the amino acid whose DNA of coding among the pPX2003 (class of meningococcus I described in the embodiment 4 pilE construction) with the suitable regional replacement (SEQ ID NO:4) of pPX2002 (construction of gonococcus pilE described in the embodiment 3 comprises 7 amino acid whose signal peptides).Except that in particular the following, adopt the flow process among the embodiment 2.

In the following manner with of the same area displacement of meningococcus pilE gene conservative 5 ' end regions with Diplococcus gonorrhoeae bacterial strain Pgh-1, following the BsmBI site is introduced in the meningococcus pilE gene: from the pPX2003PCR DNA amplification, adopt following primer: 5 ' CCG GCG CGT CTC TCA CGG CGAATG GCC CGG C3 ' is (NMRPILE) (SEQ ID NO:14) and Taq archaeal dna polymerase of (SEQ ID NO:13) and 5 ' GGG CCT GGA TCCGAG TGG CCG TGG AAA ATC ACT TAC CGC3 ' (CL-1ESPF).The PCR DNA product of expection directly is cloned among the pCR2.1 (Invitrogen) and is transformed into TOP10F ' cell, the plasmid called after pZ578 that obtains.Then, gonococcus pilE is introduced in the BsmBI site, method is as follows.The primer is (TRCUPFW) (PILEESPR) (SEQ ID NO:16) and a Pfu archaeal dna polymerase of (SEQ ID NO:15) and 5 ' GCC GCG CGT CTC CCG TGA TTC AGGTAA TAC TCG G3 ' of 5 ' GCA TAA TTC GTG TCG CTC AAG GCG C3 '.5 ' end of pcr amplification pPX2000pilE gene.Gonococcus PCR product and pZ578 with gained digests with BsmBI then, and couples together.The DNA that connects of pcr amplification again adopts (TRCUPFW) (NMRPILE) (SEQ ID NO:18) primer of (SEQ ID NO:17) and 5 ' GGG CCT GGA TCCGAG TGG CCG TGG AAA ATC ACT TAC CGC3 ' of 5 ' GCA TAA TTC GTG TCGCTC AAG GCG C3 '.

DNA PCR product has the size (about 850bp) of expectation, with NcoI and BamHI digestion, obtains the fragment of about 600bp.This fragment of gel separation directly is cloned in the pPX2000 carrier of NcoI and BamHI incision, displacement gonococcus pilE gene.The plasmid that obtains is an amicillin resistance, and mark pPX2004 is used to transform TOP10F '.This transformant analysis shows the chimeric pilE DNA that has expectation.After inducing with IPTG, compare with the meningococcus recombinant pilin amount that pPX2003 expresses, the amount of the chimeric I class of the meningococcus recombinant pilin that construction is expressed significantly increases.Extract and as purification scheme (TMAE Fractogel as described in the embodiment 7 with 1% octyl-β-D-glucopyranoside (OG) ^TMPost, 10mM Tris ^TM, pH8.5 contains 0.1% (w/v) Zwittergent ^TM3-14) purification of Recombinant gonococcus pilin obtains the chimeric I class of highly purified meningococcus recombinant pilin (yield is about 5mg/g wet cell weight).When analyzing with SDS-PAGE and laser intensity instrument, the purity of this material is greater than 90%.SDS-PAGE shows a main band of about 15,000 dalton's sizes.The length of the chimeric I class of meningococcus recombinant pilin also is 167 amino acid, comprises 7 amino acid whose signal sequences, shows as N-terminal 36 residues order-checking of this purifying protein gained.

Embodiment 6

In intestinal bacteria, make up and express gonococcus and the chimeric pilE of meningococcus II class

The initial stage clone of meningococcus II class pilE comprises from the Neisseria meningitidis bacterial strain FAM18 cellular segregation chromosomal DNA of the hair that carries disease germs and the II class pilE DNA that increases the PCR reaction.The primer of the complete pilin of following identification (comprising leader sequence) 3 ' and 5 ' end is adopted in the amplification of II class pilE gene: 5 ' GCGGCC GCC ATG GAA GCA ATC CAA AAA GGT TTC ACC C3 ' is (SEQ ID NO:19) and 5 ' GCG GCC GGA TCC GGT CAT TGT CCT TAT TTG GTGCGG C3 ' (PILE2REV) (SEQ ID NO:23) (PILE2FWD).With the strategy similar to embodiment 2, the PCR product that obtains contains a NcoI site at the section start of pilE coding region, contains a BamHI site endways.Consider for the clone, this gene is introduced in the NcoI site.This causes second amino acid whose change of signal sequence, becomes L-glutamic acid (GAA) from Methionin (AAA).As previously mentioned, estimate this variation to antigenicity or immunogenicity without any effect.The PCR product cloning in the pCR2.1 cloning vector (Invitrogen), is connected and is transformed among the intestinal bacteria TOP10F '.Screen bacterium colony containing the dull and stereotyped of 100 μ g/mL penbritins or contain on the flat board of 50 μ g/mL kantlex.From the overnight culture of these transformant, isolate plasmid DNA, and analyze with enzyme EcoRI restriction enzyme.

Clone #8, called after pPX8001 is as the source of pilE gene.The plasmid DNA of pPX8001 and pTrcHisC (Invitrogen) each with NcoI and BamHI digestion with restriction enzyme, the dna fragmentation of gel separation gained connects and is transformed among the intestinal bacteria TOP 10F '.Behind the screening amicillin resistance bacterium colony, separate the plasmid DNA of new transformant, carry out the DNA restriction analysis with BamHI and NcoI restriction enzyme.Two clones with correct unrestricted model are made dna sequence analysis.These two clones have correct dna sequence dna, called after pPX8002.

In order to measure the expression of reorganization II class pilin, 10mL is contained these clones' culture, pPX8002 grows in the 50mL pipe, and SOB wherein contains 100 μ g/mL penbritins and 12 μ g/mL tsiklomitsins, up to A ₆₀₀=1.0.The IPTG that adds final concentration 1mM has induced Recombinant Protein Expression, cultivates and continues 3 hours.When being separated with SDS-PAGE by the full cell lysate of inducing cell and using Coomassie blue stain, do not measure the band of new (inducing).The level of this prompting FAM18 pilE gene product expression is lower than the level of those Coomassie blues (dyeing) identification.When FAM18 pilE is cloned into the pET17b plasmid and be transformed into e. coli bl21 (DE3) pLysS (have or do not have pilE signal sequence), do not measure the obvious expression of recombinant protein.When the II class pilE of two other bacterial strains of Neisseria meningitidis (NmB, 2996) gene clone is gone in identical pTrcHis plasmid and the TOP10F ' expression system, obtain similar result.

Determine,, also measure bacterial strain NMB and 2996 and express II class pilin according to PCR and sequencing data.In the separating reaction as template,, adopt the primer (NMFPILE and NMRPILE) of I class and the primer (PILE2FWD and PILE2REV) of II class from some Neisseria meningitidis bacterial strain amplification pilE genes with chromosomal DNA or cell.The PCR product cloning is gone into pTrcHisC and order-checking, or directly order-checking.Carried out the sequence contrast, the sequence of similar H44/76 bacterial strain is decided to be the I class, and the sequence that is similar to the FAM18 bacterial strain is decided to be the II class.Be not to obtain sequence from all amplification bacterial strains.Carried out preliminary classification according to the PCR data.To be those obtain the bacterial strain of correct big or small PCR product with I class primer rather than with II class primer to I class bacterial strain, and II class bacterial strain is those with II class primer rather than obtains the bacterial strain of correct big or small PCR product with I class primer.

According to the test of meningococcus I class pilin, replace with the respective regions of gonococcus bacterial strain Pgh3-1 in the zone of preceding 60 amino acid of the II class of will encoding pilE (conservative aminoterminal zone).The expression of the chimeric pilE of gained is studied in a large amount of escherichia coli expression bacterial strains with various promotor.Experimental strain comprises: PR13 (RNA defective type), BL21 (protease-deficient), KS474 (periplasm protein deficient), AD494 (Novagen, it allows to form disulfide linkage in kytoplasm) and three bacterial strains of TOPP (Stratagene can be used for the non-K-21 bacterial strain of difficult expressing protein).In all experiments, in the SDS-PAGE of Coomassie blue stain, do not measure recombinant protein.Study another expression plasmid pET17b (comprising a T7 promotor) and obtained similar result.

It should be noted that the natural II class ilE gene order among the pPX8002 ends at base 447.Apart from the dna sequence dna in natural meningococcus II class pilE termination site downstream (3 '), Nucleotide 447 to 519 contains reverse tumor-necrosis factor glycoproteins (it may form stem and ring structure).Because stem and ring structure may be effective terminators of transcribing, so should additional lacking of 3 ' sequence (74 base) may influence chimeric II class pilE information transcribing in intestinal bacteria among the hypothesis pPX8002.So all clones afterwards recover this downstream 3 ' end sequence.

Use the respective regions system of gonococcus bacterial strain Pgh3-1pilE gene to replace the various piece of meningococcus bacterial strain II class pilE gene, purpose is to identify the zone that suppresses expression.Replacement areas is since 5 ' or 3 ' end, and expansion gradually.Also made up the two-way replacement of 5 ' and 3 ' end, up to the interior region of only residual following 84 Nucleotide of natural pilE II class.This zone is also replaced, and the result has rebuild rGC, and as was expected should the clone with similar in appearance to the Coomassie blue stain horizontal expression of pPX2000 recombinant pilin.FAM18 pilE gene with respective regions (bracket in the list) displacement of lower area (number listing) with Pgh3-1 with Nucleotide: single regional replacement be 1-108 (1-108), 1-181 (1-181), 1-294 (1-282), 439-499 (478-553), 379-519 (367-553), 295-519 (283-553), 295-378 (283-366); The dual area displacement is 1-294 (1-282) ﹠amp; 379-519 (367-553), 1-181 (1-181) ﹠amp; 439-499 (478-553), 1-181 (1-181) ﹠amp; 379-519 (367-553), 1-294 (1-282) ﹠amp; 439-499 (478-553).

When expressing these constructions with the pTrcHis expression system in intestinal bacteria TOP10F ', the protein level that two constructions produce can detect with Coomassie blue: first contains the Nucleotide 379-519 displacement of (comprising two sulphur rings and 3 ' overhang); Second displacement that contains Nucleotide 1-181 (comprising conservative 5 ' zone) and 379-519 (comprising two sulphur rings and 3 ' overhang).Can not cause Recombinant Protein Expression because only replace 5 ' zone, also, select this construction (Nucleotide 379-519) and be used for further research because first construction has kept the major part of natural meningococcus pilin sequence.Because meningococcus is obviously different in this zone with the proteic aminoacid sequence of gonococcus, has file (17,18) to point out that this two sulphur ring has experienced significant antigenic variation.So any immunne response at this zone (i.e. two sulphur rings) will show cross reactivity minimum between the meningococcus bacterial strain.Because the gonococcus inset is the twice (39 residues are to 18 residues) of meningococcus two sulphur ring sizes, the apparent molecular weight that the chimeric protein of generation moves on the SDS-PAGE gel is about 19,000 dalton.

The construction process of this mosaic gene is as follows.Amplification pPX8002 (FAM18II class pilE) obtains 5 ' fragment, adopts following primer: 5 ' GCG GCC GCC ATG GAA GCA ATC CAA AAA GGT TTCACC C3 ' is (SEQ ID NO:19) and 5 ' GCC GCG CGT CTC CGA ACCGGA GTT TTG TTT GCC3 ' (REV-CYS) (SEQ ID NO:20) (PILE2FWD).Obtain gonococcus two sulphur rings (being 3 ' end of gonococcus gene) from pPX2000 amplification, adopt primer 5 ' CCG GGC CGT CTCGGT TCG GTA AAA TGG TTC TGC3 ' (FWD-CYS) (SEQ ID NO:21) and 5 ' GGG CCT GGA TCC GTG GGA AAT CAC TTA CCG3 ' (PILEREV) (SEQ ID NO:22).The PCR product that obtains of purifying with restriction enzyme BsmBI digestion, then is connected to form the chimeric pilE of total length respectively, again with primer PILE2FWD and PILEREV amplification.This PCR product digests with restriction enzyme NcoI and BamHI, connects into the pTrcHisC carrier of a similar restriction, is transformed in the TOP10F ' competent cell.

Cultivate transformant, and analyze with restriction enzyme NcoI and BamHI.Four clones have been analyzed with correct big or small inset with restriction enzyme StuI.Wherein, three have correct restriction figure.Measured and had among three clones of correct unrestricted model two sequence.Clone #5 has correct dna sequence dna, called after pPX8017.The nucleotide sequence that this clone contains is listed among the SEQ ID NO:3, wherein Nucleotide 1-378 from Neisseria meningitidis and Nucleotide 379-510 from gonococcus.

In the 50mL pipe, checked expression with the 10mL substratum.Cell is grown (this SOB is added with 100 μ g/ml penbritins and 12 μ g/ml tsiklomitsins) up to A in SOB ₆₀₀Be about 1.0.Adding final concentration is the IPTG inducing culture thing of 1mM.Allow growth continue 3-4 hour, collect the cell of this time point then by centrifugal (13,689 * g, 20 minutes, 4 ℃) ,-20 ℃ of preservations.For fermentor tank, inoculate the bottle that shakes that contains the 500mL substratum with dull and stereotyped overnight culture or freezing storage liquid, regrowth is spent the night.With this liquid culture inoculate into the Biostat B fermentor tank that contains the 8.9L substratum (Braun Biotech, Allentown, PA) in.When with final concentration being the additional HySoy that contains of 1% (w/v) glucose ^TMSubstratum the time, bacterial growth in the fermentor tank improves.When culture reaches A ₆₀₀During=4.0-6.0, adding final concentration is the IPTG of 1mM, allows the long 2-4 of cell regeneration hour centrifugal then (13,689 * g, 20 minutes, 4 ℃) collecting cell.Abandon nutrient solution, cell precipitation-20 ℃ preservation.By inducing of IPTG, the increase of chimeric II class recombinant pilin is very significant.

In the presence of sodium laurylsulfonate (SDS-PAGE), analyzed derivative culture samples with polyacrylamide gel electrophoresis.Can be observed the chimeric II class of reorganization meningococcus pilin (apparent molecular weight about 19 with Coomassie blue stain, 000 dalton), it identifies by using and makes the Western trace at the polyclonal antiserum of a kind of gonococcus peptide (Glu Ala Ile Leu LeuAla Glu Gly Gln Lys Ser Ala Val Thr Glu Tyr Tyr Leu Asn His Gly Lys) (SEQ ID NO:24) and obtain proof that this peptide is positioned at the N-terminal conservative region of II class pilin.The chimeric II class of meningococcus recombinant pilin length is 170 amino acid (comprising signal peptide, SEQ ID NO:4), and wherein amino acid/11-126 is from Neisseria meningitidis, and amino acid/11 27-170 is from gonococcus.

Embodiment 7

Separate and purification of Recombinant gonococcus pilin from intestinal bacteria

With the reorganization gonococcus pilin that obtains in following step purifying the foregoing description 2 and 3.This step can be used for the meningococcus recombinant pilin that obtains among the purifying embodiment 4 equally, and can be used for the chimeric I class of the meningococcus recombinant pilin that obtains among the preliminary purification embodiment 5.Subsequently, described in following embodiment 8, improved the separating step of the chimeric I class of meningococcus recombinant pilin.The colibacillary subcellular fractionation of express recombinant pilin separates and show that this protein binding more may be an inner membrance, because 1% (v/v) Triton on cytolemma ^TMX-100 can dissolve this albumen.When having attempted there is 0.05-0.1% (v/v) Triton ^TMWhen removing the e. coli protein that depollutes during X-100, recombinant pilin can not be firmly bonded on the ion exchange column when finding to be lower than pH9.5.So, checked the ability of a large amount of stain removers selective dissolution recombinant pilin from the escherichia coli membrane prepared product.

The cell precipitation that will obtain from the 1L culture (about 5g weight in wet base) is by adding 30mL 10mM Hepes (pH7.2) (Research Organics, Cleveland, OH) and 1mM EDTA it is melted, with Microfluidizer cell homogenates device (Microfluidics International Corp., Newton, MA) lysing cell.Centrifugal (12,000 * g, 10 minutes) clarification lysate and precipitation membrane (288,652 * g 1 hour).Costal fold is suspended from 33mL 10mM Hepes (pH7.4), 1mM MgCl ₂In and in order to a kind of extracting at room temperature of following stain remover 1 hour: (a) Triton ^TMX-100 (Calbiochem-Novabiochem International, SanDiego, CA), (b) reductive Triton ^TMX-100 (Calbiochem), (c) octyl-β-D-glucopyranoside (OG) (Calbiochem), (d) Zwittergent ^TM3-8 (Z3-8) (Calbiochem), (e) Zwittergent ^TM3-10 (Z3-10) (Calbiochem), (f) Zwittergent ^TM3-12 (Z3-12) (Calbiochem), (g) Zwittergent ^TM3-14 (Z3-14) (Calbiochem), (h) Empigen BB ^TM(Calbiochem) or (i) Tween ^TM80 (ICN, Cleveland, OH).

Empigen BB ^TM(1%v/v), Zwittergent ^TM3-10 (1%w/v), reductive Triton ^TMX-100 (1%v/v), have Zwittergent ^TMThe octyl glucoside (1%v/v) of 3-10 (1%w/v) or 3-14 (0.1%w/v) selective extraction has respectively obtained recombinant pilin (containing the most micro-e. coli protein pollutes).Zwittergent ^TM3-12 is even 0.1% (w/v) dissolved recombinant pilin and a large amount of e. coli proteins.Triton ^TM80 the test any one concentration all can not extract recombinant protein (0.1-1%v/v).

By centrifugal (288,652 * g 1 hour), from undissolved membrane substance, isolate dissolved albumen.Supernatant liquor (containing recombinant pilin) is 4 ℃ of dialysed overnight, dialyzate 10mM Tris ^TM(pH8.5) contain a kind of of following nonionic detergent: (a) 0.1% (w/v) Zwittergent ^TM3-14, (b) 1% (w/v) Zwittergent ^TM3-10 or (c) 1% (w/v) OG.With the dialysis material at Fractogel ^TM(RI) column fractionation separates EMD TMAE-650 (S) for EM Separations Technology, Wakefield, and this post has been used 10mMTris ^TM(pH8.5) and each stain remover balance.With contain suitable stain remover with 10mM Tris ^TM(pH8.5), the NaCl linear gradient 0-0.2M that joins comes the albumen of elution of bound.Mix the component, purity assay and the protein content that contain recombinant pilin.In order to increase the purity of recombinant pilin, the blended material is dialysed with initial damping fluid once in a while, and on the TAME post fractional separation for the second time.

The recombinant pilin of selective elution from the post, the SDS-PAGE by Coomassie blue stain makes the laser intensity assay, is highly purified (＞90% even matter).When with 1% (w/v) Zwittergent ^TM3-10,1% (w/v) OG or 0.1% (w/v) Zwittergent ^TMWhen 3-14 extracts with column chromatography for separation, obtain similar result.The yield of recombinant pilin (being the major portion of e. coli protein total amount) is about 10mg/L in the culture that shakes the bottle growth with SOB substratum 1.5L.As recombination bacillus coli (containing pPX2002) HySoy ^TMWhen growing in fermentor tank for the substratum on basis, the yield of the recombinant pilin of purifying is increased to about 30mg/L culture, corresponding to every gram cell quality 7mg recombinant pilin.When containing 1% glucose in the fermentor tank, the total recovery of recombinant pilin is increased to about 100mg/L.

The recombinant pilin of purifying (PBS) dialyse with the 10mM sodium phosphate, the 140mMNaCl (pH7) that contain 0.05% (w/v) Z3-14, sterile filtration, 4 ℃ of preservations or-20 ℃ are frozen.

Embodiment 8

Separate and the chimeric I class of purifying meningococcus recombinant pilin from intestinal bacteria

As described in embodiment 2, large scale culturing contains the Bacillus coli cells of pPX2004 and grows in Biostat B fermentor tank.(about 88 gram weight in wet base intestinal bacteria pPX2004) are resuspended among 440mL 10mMHepes, the 1mM EDTA (pH7.5) with bacterial cell, and (Microfluidics Corp., Newton MA) breaks with Microfluidizer Model 110Y.The disruptive cell is by centrifugal (6,084 * g, 20 minutes, 10 ℃) clarification.Collect supernatant liquor, by centrifugal (205,471 * g, 1 hour, 10 ℃) separatory membrane component.Precipitation is resuspended to 220mL 10mM Hepes, 1mM MgCl with homogenizer ₂, in 1% (w/v) octyl-β-D-glucopyranoside (pH 7.5), and stirring at room 90 minutes.Centrifugal suspension (205,471 * g, 1 hour, 10 ℃).After centrifugal, the supernatant liquor that will contain the chimeric I class of dissolved recombinant pilin is by 0.22 μ Nalgene vacuum filtration film, 4 ℃ of preservations.The pH that regulates octyl glucoside extract with dense NaOH is to pH 8.5, and 200mL TMAE Fractogel then packs into ^TM(NJ), this post has been used 25mM Tris for EM Separations Technology, Gibbstown on the post ^TM, 0.1% (w/v) Zwittergent ^TM3-14 (pH8.5) balance.With the 400mL level pad of adding uncombined albumen of flush away from the post.The wash-out 25mMTris of recombinant pilin ^TM, 0.1% (w/v) Zwittergent ^TM3-14 (pH8.5), the linear gradient NaCl of 10 times of column volumes of joining (0-0.2M NaCl), flow velocity 10.0mL/ minute.Mix the component that contains chimeric I class recombinant pilin, use dH ₂O was with dilution in 1: 1, and packing into, (Bio-Rad, Hercules CA) (have used 10mM NaPO to 100mL 40 μ m ceramic hydroxyl phosphatic rock posts ₄, 0.1% (w/v) Zwittergent ^TM3-14 (pH6) balance).With the 200mL level pad of adding uncombined albumen of flush away from the post.The wash-out of chimeric I class recombinant pilin is with containing 0.1% (w/v) Zwittergent ^TM10 times of column volume linear gradient NaPO of 3-14 ₄(10-150mM NaPO ₄) liquid, flow velocity 5.0mL/ minute.With SDS-PAGE Analysis and Screening each component, mix the component that those contain chimeric I class recombinant pilin.With the gel of laser intensity instrument detection Coomassie blue stain, the purity of measuring purifying substance is at least 95%.The yield of the chimeric I class recombinant pilin of purifying is about 35mg/g weight in wet base cell.

Embodiment 9

Separate and the chimeric II class of purifying meningococcus recombinant pilin from intestinal bacteria

Unless otherwise indicated, all steps are all carried out at room temperature.The refrigerated Bacillus coli cells is resuspended to 10ml 10mM Hepes (pH7.2), and 1mM EDTA/g cell is with Microfluidizer cell homogenates device homogenate ruptured cell.Cell lysate is by centrifugal (13,689 * g, 30 minutes) clarification.The supernatant liquor that obtains continues centrifugal (388,024 * g, 30 minutes, 4 ℃).Abandon supernatant liquor, will contain the precipitation-20 ℃ freeze overnight of film.Film precipitation 9mL/ pipe is resuspended to 10mM Hepes (pH 7.2), 1mM MgCl ₂In, with 1% (w/v) Zwittergent ^TM3-16 (Calbiochem) extracted 1 hour.Centrifugal suspension (388,024 * g, 30 minutes) is used above-mentioned Zwittergent again ^TM3-16 extracts the precipitation that obtains.After centrifugal (388,024 * g, 30 minutes), with pellet resuspended in 9mL 50mM Tris ^TM(pH8.0), among the 5mM EDTA, extract with 1% (w/v) N-lauryl sarcosyl (Sigma) room temperature spend the night (slight vibration).This causes the dissolving of the chimeric II class of meningococcus recombinant pilin.Remove insoluble substance by centrifugal (388,024 * g, 30 minutes), abandon it.Adding final concentration in the supernatant liquor that contains the chimeric II class of meningococcus recombinant pilin is the Zwittergent of 1% (w/v) ^TM3-14, and use 50mM Tris ^TM(pH8.0), 10mM EDTA, 1%Zwittergent ^TM3-14 this material of dialysing spends the night.Get equal portions (1mL, 0.3mg albumen) dialysate and pass through MonoQ ^TM(NJ) (5 * 10mm), this post has been used 50mM Tris to post for Pharmacia, Piscataway ^TM(pH8.0), 10mM EDTA, 1%Zwittergent ^TM3-14 balance, flow velocity are 0.5mL/ minute.Merge and contain chimeric II class recombinant pilin debond material, use 10mM NaPO ₄(pH6.8), 1% (w/v) Zwittergent ^TMThe 3-14 dialysed overnight.The purity of this material is about 80%, is used for following embodiment 26 and 27 described researchs.This material is further purified by 1mL hydroxyapatite column (Bio-Rad), and this post has been used 10mMNaPO ₄(pH6.8), 1% (w/v) Zwittergent ^TMThe 3-14 balance.With containing 1% (w/v) Zwittergent ^TMThe linear gradient 0-0.5M NaPO of 3-14 ₄The chimeric II proteinoid of the meningococcus of liquid wash-out purifying.With the chimeric II class of SDS-PAGE screening each component recombinant pilin (gel dyes with Coomassie blue or silver).Two kinds of analyses all show, have single polypeptide band, and its molecular weight is about 19,000 dalton.Analyze polyacrylamide gel by laser intensity and show that the purity of this material is greater than 95%.

Embodiment 10

The analytical procedure of gonococcus recombinant pilin

(Pierce, Rockford IL) measure protein content, are standard with BSA with the test of two quinic acid albumen.The purity of protein Preparation thing with coomassie brilliant blue staining polyacrylamide gel (electrophoresis when having SDS) (SDS-PAGE) and is carried out the laser intensity analysis and is determined with Personal Densitometer SI (Molecular Devices).The evaluation of pilin confirms that by the Western trace this monoclonal antibody is (Biospacific) at the purifying pili of gonococcus bacterial strain P9 with embodiment 2 described monoclonal antibodies in the goods.The N terminal sequence of pilin is measured with Applied Biosystems 477A protein sequencing.When the recombinant pilin to purifying carries out the order-checking of N end, often measure two sequences.The main complete pilin of sequence representative comprises 7 amino acid leader sequences.The secondary sequence of forming the 10-20% sample is a recombinant pilin, and it has lacked leader sequence, and sequence is from phenylalanine (the N end residue of ripe gonococcus pilin).For these two kinds of recombinant pilin forms, the result that the order-checking of aminoterminal residue provides is consistent with the sequence of inferring from dna sequence dna.

With Finnagan MAT Lasermat ^TM2000 (San Jose CA), with the auxiliary laser desorption ionization of matrix flight time (MALDI-TOF) mass spectrometry, has measured the quality of recombinant pilin.With h-Mb calibrate this instrument to its desired qualities (16,951.5 dalton) 0.01% in.Recombinant pilin and isopyknic cyano group-4-hydroxycinnamic acid matrix (10mg/mL, 70: 30 ethyl ureas: in 0.1% (v/v) trifluoroacetic acid/water) is mixed.(1 μ L) is stored in the sample phase with this mixed solution of portion, and be air-dry, does the MALDI-TOF mass spectroscopy then.The average data of analyzing (the each analysis is the summation of 10 material feedings) for 15 times are determined the quality of recombinant pilin.Recombinant pilin (band signal peptide) molecular weight of measuring is 18,000 dalton, and is suitable with the molecular weight of estimating according to aminoacid component (17,981).Every batch all measures the small peak that quality is 17,232 dalton (on average).The difference of two kinds of form molecular weight of recombinant pilin (769 dalton) is losing of the first six amino acid of leader sequence (Met Asp Thr Leu Gln Lys) (SEQ ID NO:2, amino acid/11-6), and its molecular weight is 774 dalton.

Use AG Superose ^TM(Pharmacia, Piscataway is UJ) (to contain 0.05% (w/v) Zwittergent for 12 posts ^TMThe PBS balance of 3-14), makes size exclusion chromatography, obtain extremely different recombinant pilin apparent molecular weights.Under these conditions, this proteic wash-out position corresponding is 68,899 in molecular weight.This points out this recombinant protein cohesion.Yet, be subjected to the influence of albumen shape to a great extent from the size-exclusion column eluted protein.The result of velocity sedimentation centrefuge experiment shows that the molecular weight of recombinant pilin in solution is about 45,000 dalton.In order to remove stain remover (Zwittergent ^TM3-14), recombinant protein is fully dialysed with PBS.The recombinant protein of dialysis shows as solvable, and does not also precipitate at high speed centrifugation (122,000 * g, 1 hour).Do not attempt proof and whether can from recombinant protein, remove stain remover fully.Analyze this material with PBS do gel-filtration, show that this proteic apparent molecular weight is 452,349 dalton.This points out it to experience further cohesion.There is not to determine the quantity of subunit in two kinds of polymers.

452,349 can only be considered as the value of an estimation, are in the micelle because albumen may remain, as do not know whether stain remover is removed fully from sample.In view of recombinant pilin has diluted about 15-30 fact doubly when being mixed with vaccine, it seems that the apparent molecular weight of the recombinant pilin in the vaccine probably is about 450kD.

Embodiment 11

Prepare immune serum from recombinant pilin

(female, 200g) the subcutaneous immunization 20 μ g gonococcus recombinant pilin that is mixed with the purifying of adjuvant has carried out Studies on Immunogenicity by giving cavy.The adjuvant of research is: (a) Stimulon that joins with PBS (pH6) ^TMQS-21 (25 μ g/ dosage); (b) aluminum phosphate of joining (Lederle Laboratories, Pearl River, NY, 100 μ g/ dosage) with PBS (pH7); Or it is (c) single with PBS (pH7).At first, animal is in the 0th, 4 and 8 all immunizations, and is clear in the blood sampling of the 0th, 4,6 and 10 weeks.The analysis of immune response time process shows that the vaccine inoculation of doing in the 8th week for the third time not enhancing immunity is replied, and therefore the research of doing with recombinant pilin afterwards ends at the blood sampling in the 6th week.

Regulate the ability of gonococcus recombinant pilin immunne response in order to study adjuvant, mouse (female, 8 one full year of life, 5 or 10 every group) is collected serum at the albumen of the 0th, 4 and 6 all subcutaneous immunization 1-10 μ g purifying in the 0th, 4,6 and 8 weeks.Carry out the vagina lavation in the 8th week, RPMI 1640 (75 μ L) is instilled into vagina and aspirates 3-4 time.Merge every group of irrigating solution, 50 μ L foetal calf serums are joined in each amalgamation liquid.

For the chimeric I class of meningococcus recombinant pilin, mouse is only accepted immunization in the 0th and 4 weeks, collects serum in the 0th, 4 and 6 weeks.In mouse, studied the parenteral immunogenicity of all recombinant pilins, studied following adjuvant: (a) Stimulon that joins with PBS (pH6) ^TMQS-21 (25 μ g/ dosage); (b) aluminum phosphate of joining (Lederle Laboratories, Pearl River, NY, 100 μ g/ dosage) with PBS (pH7); (c) MPL that joins with PBS (pH7) ^TMAluminum phosphate that (50 μ g/ dosage) (d) joins with PBS (pH7) (100 μ g/ dosage) and MPL ^TM(50 μ g/ dosage) or single PBS (pH7) that uses.

For the chimeric II class of meningococcus recombinant pilin, the Stimulon that the 0th and 4 all cavys are accepted 20 μ g albumen and join with PBS (pH6) ^TMQS-21 (25 μ g/ dosage) adjuvant is collected serum in the 0th, 4 and 6 weeks.

Assessed capability approach that recombinant expressed pili (the chimeric I class of gonococcus or meningococcus) mucosa immunity-inducing replys and be the salt solution (have or do not have the natural Toxins,exo-, cholera of 1 μ g) that immunization (a) 2.5 μ L in the mouse nose is contained 1 or 10 μ g gonococcus recombinant pilins, or (b) the chimeric I class of 5 μ g recombinant pilin is diluted in 10 μ LPBS (pH7), there are or do not have 1 μ g mutant CT-CRM, E29H Toxins,exo-, cholera.Give immunization in the 0th, 2 and 3 weeks.

Embodiment 12

The Western engram analysis of anti-gonococcus recombinant pilin immunne response

According to embodiment 11 described schemes, with the gonococcus recombinant pilin immunization cavy of purifying.At first with the Western engram analysis immunization cross the antiserum(antisera) of the cavy of gonococcus recombinant pilin, (not having display data).These traces show that the antiserum(antisera) of anti-gonococcus recombinant pilin discerned a band corresponding to pilin in mao full cell lysate of gonococcus cell that carries disease germs; Band does not dye in the aseptic hair cell lysate of same gonococcus bacterial strain.

From the antiserum(antisera) of the cavy of gonococcus pilin oligomer immunization, obtained to compare data.Obtained the pilin oligomer as above-mentioned (4) by the complete pili that dissociates.Briefly, this comprise with complete pili in 37mM sodium phosphate (pH12) 4 ℃ the dialysis 48 hours, use 50mM Tris then ^TM, 145mMNaCl (pH8.0) dialysis, centrifugal then (100,000 * g, 1 hour) clarification pilin oligomer.After centrifugal, the pilin oligomer is retained in the supernatant liquor.Compare with the antiserum(antisera) of anti-gonococcus recombinant pilin, pilin oligomer antiserum(antisera) can combine with the cell lysate of the hair that carries disease germs, simultaneously also many other bands (not having display data) of the combined belt and the hair cell lysate that do not carry disease germs.These bands have been represented the pollutent in the pilin oligomer goods, infer that they do not combine with pili.

Embodiment 13

Reorganization gonococcus pilin ELISA

Measured the terminal point titre of the albumen or the bacterial cell of purifying by ELISA.In all ELISA operations, unless otherwise indicated, all cultivated at room temperature 1 hour.The terminal point titre is defined as the extrapolation extent of dilution of light absorption value than blank well (not containing first antibody) big 0.10.For the guinea pig antiserum analysis, the recombinant pilin of purifying is diluted in 0.1M Tris ^TM(pH8), final concentration is 1 μ g/mL.Every part (100 μ L) joins in the hole of microtiter plate (Immulon II, Nunc, Naperville IL), 4 ℃ of overnight incubation.With Skanwash 300 titer plate washers (Skatron Instruments, Alexandria, VA), to contain 0.05% (v/v) Tween ^TMThe PBS washing of-20 (PBS-T) 5 times.Each seals all holes with the PBS-T that 200 μ L contain 1% (w/v) BSA, washing, and each hole adds a antiserum(antisera) (being diluted in the PBS-T that contains 0.1% (w/v) BSA).The washing titer plate, with 100 μ L couplings the alkaline phosphatase of the anti-cavy IgG of rabbit (heavy chain or light chain) detect bonded first antibody (Zymed Laboratories, South San Francisco, CA), this conjugate is diluted in the 50mM Tris that contains 0.1% (w/v) BSA at 1: 2000 ^TM(pH8) in.The washing titer plate, with 100 μ L/ hole phosphoric acid p-nitrophenyls (Sigma) (2mg/mL in the 0.5M diethanolamine, 0.25mM MgCl ₂, pH9.8) develop the color.After 30 minutes, add 50 μ L 3N NaOH termination reactions.(MolecularDevices, Sunnyvale CA) read the 405nm absorbancy with Thermomax ELISA plate reader.

The animal of useful recombinant pilin immunization good responsing reaction is all arranged, show as table 1 with antigen ELISA.

Table 1

The reorganization gonococcus pilin bonded of blended anti-gonococcus recombinant pilin guinea pig antiserum and purifying

The terminal point titre ^*

Prepared product	Immunogen	The terminal point titre
		The terminal point titre			The 0th week	The 4th week	The 6th week
		1	Reorganization Pgh3-1 pilin (goods 1)	≤100	The 0th week	The 4th week	The 6th week	51,345	494,805
2	Reorganization Pgh3-1 pilin (goods 2)	1	Reorganization Pgh3-1 pilin (goods 1)	≤100	54	30,237	594,298	51,345	494,805
2	Reorganization Pgh3-1 pilin (goods 2)	3	Reorganization Pgh3-1 pilin (goods 3)	≤100	54	30,237	594,298	24,830	546,682

^*Three batches of different recombinant pilins are as immunogen.Cavy is in the 0th and 4 all immunizations (subcutaneous injection), and in the blood sampling of the 0th, 4 and 6 weeks.Blended serum is analyzed.

Adjuvant is to the influence of immunne response

In mouse, studied of the influence of following adjuvant: (a) Stimulon that joins with PBS (pH6) to gonococcus recombinant pilin immunne response ^TMQS-21; (b) aluminum phosphate of joining with PBS (pH7); (c) MPL that joins with PBS (pH7) ^TM(d) aluminum phosphate and the MPL that joins with PBS (pH7) ^TMOr it is single with PBS (pH7).In order to analyze mouse resisting anteserum, the following antigen ELISA flow process of having improved.(Cambridge's microtiter plate MA) is spent the night with the PBS37 ℃ of bag that 100 μ L contain 1 μ g/mL recombinant pilin for Costar EIA/RIA, Coming Costar.With Skantron 300 titer plate washers to contain 0.1% (v/v) Tween ^TM-20 PBS wash plate 5 times.With containing 0.1% (w/v) gelatin and 0.02%NaN ₃PBS seal each hole.First antibody dilutes with PBS, and this PBS contains 0.1% (w/v) gelatin, 0.05% (v/v) Tween ^TM-20 (PBS-TG) and 0.02% (w/v) NaN ₃, every part 100 μ L cultivated in microtiter plate 2 hours.After the washing, (Brookwood Biomedical, Birmingham is AL) with PBS-TG and 0.02% (w/v) NaN with the anti-mouse IgG of biotinylated rabbit (Fc zone) ₃Dilution in 1: 8000 detects first antibody.Washing is dull and stereotyped, is diluted in PBS-TG and 0.02% (w/v) NaN at 1: 5000 in order to Streptavidin link coupled horseradish peroxidase (Zymed Laboratories) successively ₃(cultivating 30 minutes) detects second antibody.Washing is dull and stereotyped, the 0.5mg/mL2 that joins with the 0.1M Citrate trianion that contains 0.03% (v/v) hydrogen peroxide (pH 4.2), 2 '-azine-two-(3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) (ABTS) developed the color 30 minutes, with SLT 340ATTC microplate reader (SLTLabinstruments, Research Triangle Park NC) monitors at 405nm.Depict data with log-log plot, as above-mentioned definite terminal point titre.

As recombinant pilin MPL ^TMDuring for adjuvant, in mouse, obtain humoral immunoresponse(HI), on the order of magnitude, be similar to and use Stimulon ^TMQS-21 (table 2A) is observed.The vaginadouche thing of analyzing same animal shows that also the vaginadouche thing of these animals has tangible IgG titre (table 2B).

Table 2A

Adjuvant is to the influence of gonococcus recombinant pilin immunne response ^*

Body fluid:

	Antigen ELISA	Full cell ELISA
		Full cell ELISA					Homologous strain	The allos bacterial strain
		Adjuvant	Recombinant pilin	Pgh3-1	LB2	#11	Homologous strain	The allos bacterial strain				T-1	I756
AlPO ₄	361,260	Adjuvant	Recombinant pilin	Pgh3-1	LB2	#11	249,410	93,444	141,622	42,535	89,615	T-1	I756
AlPO ₄	361,260	MPL ^TM	1,262,735	902,891	499,223	586,642	249,410	93,444	141,622	42,535	89,615	165,301	321,340
AlPO ₄+M PL ^TM	1,159,670	MPL ^TM	1,262,735	902,891	499,223	586,642	265.269	112,856	160,814	41,368	110,162	165,301	321,340
AlPO ₄+M PL ^TM	1,159,670	StimulonT MQS-21	4,915,200	1,053,446	478,338	440,014	265.269	112,856	160,814	41,368	110,162	111.753	333,341

Table 2B

Vaginal douche ^*

Adjuvant	The recombinant pilin titre		Full cell titer ^***
	The recombinant pilin titre		Full cell titer ^***		IgG	IgA	LB2P+	LB2P-
	AlPO ₄	1,452	262	68	IgG	IgA	LB2P+	LB2P-	≤50
MPL ^TM	AlPO ₄	1,452	262	68	6,409	26	1,128	0	≤50
MPL ^TM	AlPO ₄+MPL ^TM	938	12	120	6,409	26	1,128	0	≤50
Stimulon ^TM QS-21	AlPO ₄+MPL ^TM	938	12	120	2,716	175	311	37	≤50

^*Balb/c mouse (5 every group) is at the 0th, 4 and 6 all immunization 10 μ g recombinant pilin, wherein adjuvants such as above-mentioned.Animal is in the blood sampling of the 0th, 4,6 and 8 weeks.Pooled serum to the 8th week performs an analysis.Terminal point titre≤5,000 in the 0th week.

^*The mixing vaginal douche that obtains in the 8th week.

^{* *}Full cell ELISA with air-dry cell.

P+: the cell of the hair that carries disease germs.P-: the aseptic chalaza variant of mao parental cell deutero-carries disease germs.

Embodiment 14

The full cell ELISA of gonococcus

In full cell ELISA, confirmed, find the cross reaction epi-position of the obvious number that recombinant pilin is expressed in the pili of complete assembling, wherein the antiserum(antisera) of the recombinant pilin of antivenom purification demonstrates the high titre to the gonococcus bacterial strain of numerous expressing heterologous pili.Guinea pig antiserum is measured with following scheme in conjunction with the ability of the gonococcus cell of living.Microtiter plate (Immunolon II) spends the night with the PBS4 ℃ of cultivation that 100 μ L contain 0.01% (w/v) poly-L-Lysine (Sigma).With the Dacron swab bacteria Agr culture in eve is gathered in the crops among the PBS, the turbidity of suspension is adjusted to A ₆₀₀=1.0.Remove the polylysine liquid in the microtiter plate, every part 100 μ L bacterial suspension is joined in the hole.With PBS wash plate 5 times, contain PBS (PBS-B) sealing of 1% (w/v) BSA with manual type Nunc washer with 200 μ L.Then, use PBS wash plate 5 times, every part 100 μ L is joined in the hole with the antiserum(antisera) that the PBS that contains 0.1% (w/v) BSA dilutes.After PBS washing 5 times, detect the bonded antiserum(antisera) with the above-mentioned suitable alkaline phosphatase enzyme conjugates of 100 μ L (being diluted among the PBS that contains 0.1%BSA) with 1: 2000.Wash plate is as above-mentioned colour developing and carry out absorbancy reading (30 minutes colour developing).

When analyzing with full cell ELISA, the guinea pig antiserum of anti-recombinant pilin is incorporated into the bacterial isolates of the hair that carries disease germs of each geographic area, but debond is in the cell (table 3) of the corresponding no pili of same gonococcus bacterial strain.

Table 3

The guinea pig antiserum of anti-recombinant pilin is in conjunction with the terminal point titre of each bacterial strain gonococcus cell alive ^*

Bacterial strain	The source	Pgh3-1 recombinant pilin (goods 1)	Pgh3-1 recombinant pilin (goods 2)	Pgh3-1 recombinant pilin (goods 3)
Bacterial strain	The source	Pgh3-1 recombinant pilin (goods 1)	Pgh3-1 recombinant pilin (goods 2)	Pgh3-1 recombinant pilin (goods 3)	Pgh3-1P+	The Pittsburgh	2,940,971	2,461,272	2,373,641
Pgh3-1P-	The Pittsburgh	＜250	＜250	＜250	Pgh3-1P+	The Pittsburgh	2,940,971	2,461,272	2,373,641
Pgh3-1P-	The Pittsburgh	＜250	＜250	＜250	LB2P+	Unknown	505,439	481,815	825,517
LB2P-	Unknown	187	34	114	LB2P+	Unknown	505,439	481,815	825,517
LB2P-	Unknown	187	34	114	I-756P+	Ontario Lake, Canada	689,309	713,904	1,026,231
1948P+	Romania	290,367	259,487	325,640	I-756P+	Ontario Lake, Canada	689,309	713,904	1,026,231
1948P+	Romania	290,367	259,487	325,640	1635P+	Panama	2,714,006	≥546,750	≥546,750
22714FB P+	Fort Bragg，NC	397,763	346,895	498,882	1635P+	Panama	2,714,006	≥546,750	≥546,750
22714FB P+	Fort Bragg，NC	397,763	346,895	498,882	T-1P+	Unknown	189,490	317,370	272,828
M-2P+	Unknown	124,906	117,734	151,966	T-1P+	Unknown	189,490	317,370	272,828
M-2P+	Unknown	124,906	117,734	151,966	3138K P+	Korea S	220,730	179,127	584,044
N-2P+	The Norfolk, VA	350,210	337,232	435,164	3138K P+	Korea S	220,730	179,127	584,044

J474B P+	Jamaica	238,640	205,247	380,970
J474B P+	Jamaica	238,640	205,247	380,970	Fresh clinical isolates 52407 P+	The Tampa, FL	598,124	890,678	778,607
#11P+	Rochester，NY	214,673	292,358	385,985	Fresh clinical isolates 52407 P+	The Tampa, FL	598,124	890,678	778,607
#11P+	Rochester，NY	214,673	292,358	385,985	2-57-U17P+	Chapel Hill，NC	1,364,126	2,379,267	3,791,263
2-42-U14P+	Chapel Hill，NC	1,449,757	1,964,641	11,619,267	2-57-U17P+	Chapel Hill，NC	1,364,126	2,379,267	3,791,263

^*Three batches of different recombinant pilins are as immunogen.As above-mentioned immunization cavy (subcutaneous irradiation) and blood sampling.Analyze the pooled serum in the 6th week.Terminal point titre≤250 of the 0th all serum.

Carry out the full cell ELISA analysis of anti-recombinant pilin (table 2) mouse resisting anteserum with microtiter plate, wherein use following scheme the bacterium complete drying in the hole.Be harvested among the PBS with the bacteria Agr culture of Dacron swab, and the turbidity of bacterial suspension is adjusted to A eve ₆₀₀=0.1.Every part of (100 μ L) bacterial suspension joins in the hole 37 ℃ of air-dry plates.After evaporating all liquid, sealing plate is saved in use in 4 ℃.The antigen ELISA flow process of above-mentioned mouse resisting anteserum is adopted in remaining test.Full cell ELISA data (table 2 and 3) prompting, recombinant pilin inductive antibodies is in mao conserved epitope on gonococcus surface that carries disease germs.

Embodiment 15

Induce the mucosal immune response of anti-gonococcus recombinant pilin

Because gonorrhoea is a kind of disease of reproductive tract mucous membrane, the mucosal immunity ability that interesting check mucosa immunity-inducing is replied.Implement as follows.In the 0th, 1 and 2 weeks, with immunized mice in gonococcus recombinant pilin salt solution (1 or 10 μ g/10 μ L) (have or do not have the natural Toxins,exo-, cholera of the 1 μ g) nose.In the 0th, 1 and 2 weeks, immunization recombinant pilin in 5 every group Swiss-Webster mouse noses (have or do not have Toxins,exo-, cholera).Blended serum is analyzed.Terminal point titre＜50 in the 0th week.

As shown in table 4, when animal is used the recombinant pilin immunization of no adjuvant, in antigen ELISA, measure significant immunne response (the 0th all titres＜300).This is replied by adding natural Toxins,exo-, cholera and is improved.

Table 4

The reorganization gonococcus pilin bonded of the mixing mouse resisting anteserum of anti-gonococcus recombinant pilin and purifying

The terminal point titre

Antigen (dosage μ g)	Adjuvant (dosage μ g)	The 22nd day	The 36th day	The 50th day
Antigen (dosage μ g)	Adjuvant (dosage μ g)	The 22nd day	The 36th day	The 50th day	Recombinant pilin (1)	Do not have	1,648	1,287	2,291

Recombinant pilin (10)	Do not have	29,046	20,658	71,067
Recombinant pilin (10)	Do not have	29,046	20,658	71,067	Recombinant pilin (1)	Toxins,exo-, cholera (1)	4,673	7,526	3,273
Recombinant pilin (10)	Toxins,exo-, cholera (1)	107,011	321,714	280,079	Recombinant pilin (1)	Toxins,exo-, cholera (1)	4,673	7,526	3,273
Recombinant pilin (10)	Toxins,exo-, cholera (1)	107,011	321,714	280,079	Do not have	Toxins,exo-, cholera (1)	＜300	＜300	＜300

Then, the ELISA with anti-gonococcus bacterial strain FA1090 checks these serum to be incorporated into complete, mao ability of gonococcus cell of carrying disease germs.As above-mentioned with these cells complete drying on microtiter plate.As shown in table 5, in only with pilin, measure low titre (the 0th all titres＜300).Adding Toxins,exo-, cholera can improve and the combining of the hair cell that carries disease germs greatly.

Table 5

The mixing mouse resisting anteserum of anti-gonococcus recombinant pilin and complete, mao gonococcus cell bonded terminal point titre of carrying disease germs

Antigen (dosage μ g)	Adjuvant (dosage μ g)	The 22nd day	The 36th day	The 50th day
Antigen (dosage μ g)	Adjuvant (dosage μ g)	The 22nd day	The 36th day	The 50th day	Recombinant pilin (1)	Do not have	1,009	852	729
Recombinant pilin (10)	Do not have	6,009	6,252	5,564	Recombinant pilin (1)	Do not have	1,009	852	729
Recombinant pilin (10)	Do not have	6,009	6,252	5,564	Recombinant pilin (1)	Toxins,exo-, cholera (1)	1,133	1,456	1,048
Recombinant pilin (10)	Toxins,exo-, cholera (1)	209,522	57,767	38,127	Recombinant pilin (1)	Toxins,exo-, cholera (1)	1,133	1,456	1,048
Recombinant pilin (10)	Toxins,exo-, cholera (1)	209,522	57,767	38,127	Do not have	Toxins,exo-, cholera (1)	239	＜500	＜500

Embodiment 16

The gonococcus immunoelectronmicroscopy

Adopt following flow process to observe combining of antiserum(antisera) and the capillary bacterium that carries disease germs.Drip to gold-plated carrying in the net 5 minutes with the recA-in the logarithmic growth later stage a point of mao gonococcus liquid culture that carries disease germs, remove excessive liquid with a filter paper.Carried net 5 minutes with the PSB-S sealing, then with containing 1% fresh gelatin (Fluka, Ronkonkoma, PBS sealing NY) 10 minutes.Then at room temperature, cultivated year net 1-60 minute with the polyclonal antiserum that is diluted in PSB-B.To carry net is immersed in to remove in the PBS-B drop and does not have bonded antibody (4 * 30 seconds).To carry net is immersed in one and is incorporated into the anti-cavy IgG of donkey (Jackson Research Labs, West Grove PA) in the Radioactive colloidal gold of 12nm (to be diluted in PBS-B at 1: 5) 30 minutes, detect the bonded first antibody.As above-mentioned, flushing is carried net 5 times on the PBS-B drop then.Then with containing 1% (v/v) glutaraldehyde (ElectronMicroscopy Sciences, Fort Washington, PBS fixed sample PA) 3 minutes, again with distilled water rinsing 5 * 1 minutes, (NY) (pH8) slight stain is 30 seconds for NanoProbes, Stony Brook with the NanoVan dye liquor.Make year net contact filter paper remove all liquid, with Zeiss 10C transmission electron microscope, at 15-75,000X checks with the 80kv acceleration voltage.

Shown in immuno-electron microscope (Figure 1A), anti-recombinant pilin antibody is along length (direction) combination of the allos pili fibril on gonococcus surface.This prompting internal antibody will be incorporated into the epi-position that those bacterium surfaces exist.

Embodiment 17

The full cell ELISA of gonococcus recombinant pilin oligomer

In order to identify the biochemistry and the immunological characteristic of those complete pili (or pilin oligomer) recombinant pilin, with the recombinant pilin of purifying phosphoric acid buffer dialysis changing into recombinant pilin oligomer by pH12.Use full cell ELISA, check is incorporated into alive, the gonococcal ability of hair of carrying disease germs by this material (recombinant pilin oligomer) inductive antiserum(antisera).As shown in table 6, induce the antiserum(antisera) of generation to compare with untreated recombinant pilin, the antiserum(antisera) of anti-recombinant pilin oligomer is in conjunction with the various mao gonococcus cells that carry disease germs low-down titre being arranged.This prompting recombinant pilin oligomer has been lost the cross reaction epi-position that is present in usually in a large number on the recombinant pilin.

Table 6

PH12 (" oligomerization ") is incorporated into the recombinant pilin guinea pig antiserum and carries disease germs mao gonococcus cell eventually

The influence of some titre ^*

Bacterial strain	Recombinant pilin	The recombinant pilin oligomer ^**
Bacterial strain	Recombinant pilin	The recombinant pilin oligomer ^**	I-756	927,564	16,903
FA-19	107,100	1,982	I-756	927,564	16,903
FA-19	107,100	1,982	FA1090(2-57-U17)	721,786	6,737
LB2	905,711	4,205	FA1090(2-57-U17)	721,786	6,737
LB2	905,711	4,205	#11	225,602	6,999
#4	288,120	8,104	#11	225,602	6,999
#4	288,120	8,104	3138K	106,315	5,429
T-1	497,987	42,162	3138K	106,315	5,429
T-1	497,987	42,162	1948	166,864	8,616
J474B	576,640	25,002	1948	166,864	8,616

^*Cavy (4 every group) is in the 0th and 4 weeks, and immunization (subcutaneous injection) 20 μ g recombinant pilin antigens are with 25 μ g Stimulon ^TMQS-21 is an adjuvant.Analyze the blended serum in the 6th week.

^*At 4 ℃ with the recombinant pilin of sodium phosphate (pH12) dialysis purifying 48 hours, then with PBS dialysis 24 hours.

Embodiment 18

To sticking the human cervical cancer 1 cell inhibiting

Because pili has mediated gonococcus and combined with the elementary of people's mucomembranous cell, suppress the epithelial ability of these bacterial adhesion so studied recombinant pilin antibody.Selected cervical cancer deutero-ME-180 cell.In addition, minimize, they are grown with the fluid suspension culture thing in order to make the gathering together of capillary bacterium of carrying disease germs in these experiments.This need be with the recA-derivative of gonococcus bacterial strain Pgh3-1 or I756, to keep the expression of pili.Growth is during 4-5 hour in liquid medium, and these recA-bacterial strains do not demonstrate significantly gathers together, and it is more easy that this translates the result.

In these experiments, with ME-180 cell inoculation 8 hole slot slide glasss (Nunc), experiment these cells on same day 80-90% are paved with.Move among the PBS (being heated to 37 ℃) with the overnight culture of swab with recA-gonococcus cell, be used to inoculate the bottle that shakes that contains liquid GC substratum, this culture medium supplemented has 0.4% (w/v) NaNCO ₃, finally reach A ₆₀₀=0.2.37 ℃ of cell cultures about 4 hours with the 120RPM shaking culture, this moment, culture reached A ₆₀₀≌ 0.8.Bacterial cell suspension is done dilution in 1: 8 with RPMI 1640, and all holes of second row, 8 hole slots and 300 μ L RPMI 1640 and foetal calf serum were cultivated 1 hour at least.Remove RPMI 1640 confining liquids, add 40 μ L antiserum(antisera)s or RPMI 1640 (no bovine serum), (≈ 8 * 10 to add the bacterial suspension of 260 μ L dilution then ⁷CFU), 37 ℃/5% (v/v) CO ₂Cultivated 1 hour.Contain of RPMI 1640 flushings of the hole slot slide glass of ME180 cell with no antibiotic.Then, the bacterium and the antiserum(antisera) mixed solution of preincubation is added on the ME180 cell 37 ℃ of incubation slide glasss 30 minutes.Remove from the cervical cell individual layer and to contain, wash all holes gently 3 times with RPMI 1640 not in conjunction with the substratum of bacterial cell.After the last flushing, remove hole slot from slide glass, cell is with methyl alcohol fixing 30-60 second, and (VWR Scientific, West Chester PA) dye with the Wright-Giemsa dye liquor.In water, after the decolouring, cover glass is overlayed on each hole.

With oil immersion lens opticmicroscope check slide glass, take the photo in the typical visual field by the people who does not know test sera.Analyze resulting picture by the people who does not know sample type equally.In addition, because mao gonococcus that carries disease germs is incorporated into epithelial monolayers with the form of gathering together, influence comes quantitative measurment by count fine flora rather than bacterium individuality so antiserum(antisera) is to gonococcus cell bonded.Measure the quantity of the capillary flora that carries disease germs with each Kong Quankong finding of ten random scannings.Determined to contain the percentage ratio difference between immune serum and the normal serum hole.Equally, this analysis is independently finished by the investigator who does not know the analytic sample kind.

Initial result shows, carry disease germs hair and aseptic hair cell are with different combining with ME180 sheet monolayer cell in conjunction with pattern.Carry disease germs two strain bacterial strains of hair of gonococcus typically are incorporated into selected epithelial cell in the sheet monolayer cell with bacterial flora collection.On the contrary, corresponding homogenic aseptic hair cell or debond (Pgh3-1) in or present low-level, dispersedly in conjunction with (I756) in epithelial monolayers.So the gathering of bacterium on epithelial monolayers is relevant with the existence of pili.

In addition, having tested anti-Pgh3-1 recombinant pilin guinea pig antiserum suppresses the bacterial strain I-756 hair cell that carries disease germs and is incorporated into the ability of ME-180 cell.The analysis of typical picture (comparison diagram 2A (the 6th week) and Fig. 2 B (the 0th week)) shows, the antibody of anti-recombinant pilin the combining of mao gonococcus and cervical epithelial cell of having suppressed significantly to carry disease germs.On the contrary, compare with normal guinea pig serum, the recombinant pilin antiserum(antisera) is to the not influence (not having display data) of combination of the aseptic hair cell of same bacterial strain.Though can not measure bacterium bonded quantity under these conditions, but by counting bonded bacterial flora quantitative analysis adhesion condition when normal or immune serum exist.Determined with this method that (comparing with normal guinea pig serum) anti-recombinant pilin antiserum(antisera) has reduced and be incorporated into epithelial bacterial flora about 60%.

Though this test does not obtain the quantifiable data that is easy to get, may underestimate sero-fast effectiveness by the estimation that the count fine flora obtains.This is because other cell surface composition (as Opa albumen) has mediated this combination, and this can not expect that list just can overcome with anti-recombinant pilin antiserum(antisera).

Embodiment 19

The analytical procedure of the chimeric I class of meningococcus recombinant pilin

With the chimeric meningococcus I of embodiment 10 described analytical class recombinant pilin.As the MALDI-TOF mass spectrograph was measured, the molecular weight subunit of the chimeric I class of meningococcus recombinant pilin was 17,659 dalton, with closely similar according to molecular weight 17,676 dalton of aminoacid component expection.As this albumen Superose ^TM12 posts are (to contain 0.05% (w/v) Zwittergent ^TMWhen the PBS balance of 3-14) doing the size exclusion chromatography analysis, the apparent molecular weight of chimeric I class recombinant pilin is 69,480 dalton.The apparent molecular weight (68,899 dalton) of the gonococcus recombinant pilin that this and the same terms are analyzed down is basic identical.

Measured the N terminal sequence of the meningococcus I class recombinant pilin of purifying with the Edman edman degradation Edman, result's (deriving from three different samples) is consistent with the protein sequence of estimating.All sequences 35 residues have been measured at least.

Embodiment 20

The chimeric I class of meningococcus recombinant pilin ELISA

With embodiment 13 and 14 described methods, antivenom purification albumen or the sero-fast terminal point titre of bacterial cell have been measured with ELISA.Pooled serum with blood sampling is respectively made ELISA.On the meningococcus cell, make full cell ELISA, this cell heat-killed (56 ℃, 60 minutes) or direct complete drying on microtiter plate.At 620nm, it is 0.1 that cell suspending liquid is diluted to the 620nm absorbancy, gets the hole that every part 100 μ L places microtiter plate.Make each plate 37 ℃ or drying at room temperature, sealing is stored in 4 ℃ up to use.The flow process that changes full cell ELISA is as follows: (1) is diluted in first and second antiserum(antisera)s and contains 0.1% (v/v) Tween ^TMAmong the PBS of-20 and 0.1% (w/v) BSA; (2) with Skanwash 300 titer plate washers to contain 0.05% (v/v) Tween ^TMDull and stereotyped 5 times of-20 PBS washing.

The cavy of useful chimeric I class recombinant pilin immunization reply well, show as antigen ELISA to see Table 7.

Table 7

The chimeric I class reorganization of the meningococcus of the chimeric I class of meningococcus recombinant pilin antiserum(antisera) and purifying bacterium

Hairless protein bonded terminal point titre ^*

Adjuvant:

Blood sampling	Stimulon ^TM QS-21	AlPO ₄	Do not have
Blood sampling	Stimulon ^TM QS-21	AlPO ₄	Do not have	The 0th week	23	12	32
The 4th week	26,607	12,067	4,829	The 0th week	23	12	32
The 4th week	26,607	12,067	4,829	The 6th week	1,519,956	327,539	302,911

^*Cavy (4 every group) is at the chimeric I class of the 0th and 4 all immunizations (subcutaneous injection) 20 μ g recombinant pilin, and wherein the adjuvant of Tian Jiaing is (a) 25 μ g Stimulon ^TMThe PBS of QS-21 (pH 6); (b) 100 μ gAlPO ₄Salt solution; Or it is (c) single with PBS (pH7).Animal is in the blood sampling of the 0th, 4 and 6 weeks.Blended serum has been adopted in all analyses.

As shown in table 8, the ELISA of the hair cell that carries disease germs observed significantly reply.

Table 8

The chimeric I class of meningococcus recombinant pilin antiserum(antisera) and meningococcus (bacterial strain H355) carries disease germs

Hair cell bonded terminal point titre ^*

Adjuvant:

Blood sampling	Stimulon ^TM QS-21	AlPO ₄	Do not have
Blood sampling	Stimulon ^TM QS-21	AlPO ₄	Do not have	The 0th week	28	19	53
The 4th week	1,293	1,052	381	The 0th week	28	19	53
The 4th week	1,293	1,052	381	The 6th week	61,497	25,477	16,467

^*The cell heat-killed.

Adjuvant is to the influence of immunne response

With embodiment 13 described methods, in mouse, studied the influence of adjuvant to the immunne response of the chimeric I class of meningococcus recombinant pilin.As shown in table 9, add Stimulon ^TMQS-21 has realized antiserum(antisera) and the most significant the replying of the chimeric I class of meningococcus recombinant pilin bonded.

Table 9

The chimeric I class of the meningococcus recombinant pilin bonded terminal point titre of the chimeric I class of meningococcus recombinant pilin antiserum(antisera) and purifying ^*

Adjuvant:

Blood sampling	Stimulon ^TM QS-21	AlPO ₄	MPL ^TM	AlPO ₄/MPL ^TM	Do not have
Blood sampling	Stimulon ^TM QS-21	AlPO ₄	MPL ^TM	AlPO ₄/MPL ^TM	Do not have	The 0th week	＜50	44	＜50	31	＜50
The 4th week	44,011	29,925	40,146	110,093	＜250	The 0th week	＜50	44	＜50	31	＜50
The 4th week	44,011	29,925	40,146	110,093	＜250	The 6th week	707,084	103,437	284,455	137,686	115, 022

^*Mouse (10 every group) is at the chimeric I class of the 0th and 4 all immunizations (subcutaneous injection) 10 μ g meningococcuss recombinant pilin, and wherein the adjuvant of Tian Jiaing is (a) 25 μ g Stimulon ^TMThe PBS of QS-21 (pH6); (b) 100 μ g AlPO ₄Salt solution; (c) 50 μ g MPL ^TMPBS (pH7); (d) 100 μ g AlPO ₄With 50 μ g MPL ^TMSalt solution; Or it is (e) single with PBS (pH7).Animal is in the blood sampling of the 0th, 4 and 6 weeks.Blended serum has been adopted in all analyses.

As shown in table 10, add Stimulon ^TMQS-21 has also realized antiserum(antisera) and meningococcus the most significant the replying of hair cell bonded of carrying disease germs.

Table 10

Hair carries disease germs for the chimeric I class of meningococcus recombinant pilin antiserum(antisera) and meningococcus (bacterial strain H355)

Cell bonded terminal point titre ^*

Adjuvant:

Blood sampling	Stimulon ^TM QS-21	AlPO ₄	MPL ^TM	AlPO ₄/ MPL ^TM	Do not have
Blood sampling	Stimulon ^TM QS-21	AlPO ₄	MPL ^TM	AlPO ₄/ MPL ^TM	Do not have	The 0th week	172	146	153	160	＜50
The 4th week	6,088	2,310	4,205	5,647	311	The 0th week	172	146	153	160	＜50
The 4th week	6,088	2,310	4,205	5,647	311	The 6th week	171,718	25,135	52,053	17,039	16,617

The cell heat-killed.

Further analyze and shown that the antiserum(antisera) of anti-chimeric I class recombinant pilin can combine with the meningococcus cell of expressing I class pilin (being II class pilin in some experiments).The result who is shown in the table 11 is the evidence of part cross reaction.

Table 11

Meningococcus chimeric I class recombinant pilin and the meningococcus hair cell bonded terminal point titre of carrying disease germs ^*

Adjuvant

Bacterial strain	The type of the pilin of expressing	The 0th day	Stimulon ^TM QS-21	AlPO ₄	MPL ^TM
Bacterial strain	The type of the pilin of expressing	The 0th day	Stimulon ^TM QS-21	AlPO ₄	MPL ^TM	H355	I	409	127,383	41,190	102,987
M982	I	217	＞36,540	＞36,540	＞36,540	H355	I	409	127,383	41,190	102,987
M982	I	217	＞36,540	＞36,540	＞36,540	CDC1521	II	988	2,602	1,345	1,768
FAM18	II	3,518	＞36,540	26,513	＞36,540	CDC1521	II	988	2,602	1,345	1,768

^*Cell is without heat-killed, and directly on microtiter plate dry (room temperature).

Embodiment 21

The chimeric I class of meningococcus recombinant pilin immunoelectronmicroscopy is observed

Following employing transmission electron microscopy has been observed chimeric meningococcus I class recombinant pilin antiserum(antisera) and meningococcus bacterial strain H355 the combining of hair cell of carrying disease germs.Carefully from meningococcus culture in eve, choose a bacterium colony with asepsis ring, place and contain the improved Franz substratum of 0.5-1.0mL [1.3g/L L-glutamic acid, 20mg/L halfcystine, 10g/L Na ₂HPO ₄7H ₂O, 90mg/L KCl, 6g/L NaCl, 2g/L yeast dialysate and replenish with glucose (4g/L), L-glutamic acid (100mg/L), diphosphothiamine (200 μ g/L) and iron nitrate (5mg/L)] the microfuge pipe in.Every part of cell suspension liquid spotting 5 is online inferior to gold-plated year, and wherein every part of cell suspending liquid has been spent 5 minutes, removes excessive liquid with filter paper.Then with the PBS room temperature fixation of bacteria cell 30 minutes that contains 4% (v/v) Paraformaldehyde 96,0.1% (v/v) glutaraldehyde.Carrying net cultivates with following material one by one: (a) PBS-B, 5 minutes; (b) the fresh gelatin PBS of 1% (w/v), 10 minutes; (c) contain the PBS of 0.2M glycine, 5 minutes.As described in the embodiment 16, detect the net that carries of sealing with the chimeric I class of meningococcemia recombinant pilin antiserum(antisera) then.As shown in Figure 3A, the chimeric I class of meningococcemia recombinant pilin antibody is along length (direction) combination of pili.On the contrary, normal serum (the 0th week) does not show has any combination (Fig. 3 B) with pili.

Embodiment 22

The carry disease germs cross reactivity of hair cell of the chimeric I class of meningococcus recombinant pilin antiserum(antisera) and gonococcus

According to the sequence similarity of meningococcus I class pilin and gonococcus pilin, in the foregoing description 4, shown the antiserum(antisera) identification of anti-gonococcus recombinant pilin and be incorporated into the meningococcus cell of the hair that carries disease germs.In the present embodiment, proved that the antiserum(antisera) of the chimeric I class of meningococcemia recombinant pilin is incorporated into the gonococcus cell of the hair that carries disease germs.Table 12 and 13 has been summed up the data of mouse and cavy experiment respectively.

Table 12

The mouse resisting anteserum of the chimeric I class of meningococcemia recombinant pilin and mao gonococcus cell bonded that carries disease germs

The terminal point titre

Antigen/adjuvant

The gonococcus bacterial strain	Meningococcus reorganization I class pilin					Gonococcus recombinant pilin+MPL ^TM
The gonococcus bacterial strain	Meningococcus reorganization I class pilin					Gonococcus recombinant pilin+MPL ^TM		Stimulon ^{T M} QS-21	AlPO ₄	MPL ^TM	AlPO ₄/M PL ^TM	Do not have
I756	4,527, 943	79,927	114,958	56,627	57,426	356,936		Stimulon ^{T M} QS-21	AlPO ₄	MPL ^TM	AlPO ₄/M PL ^TM	Do not have
I756	4,527, 943	79,927	114,958	56,627	57,426	356,936	FA1090	531,627	40,406	97,224	31,267	38,122	219,600

Table 13

The guinea pig antiserum of the chimeric I class of meningococcemia recombinant pilin and mao gonococcus cell bonded that carries disease germs

The terminal point titre

Antigen/adjuvant

The gonococcus bacterial strain	Meningococcus reorganization I class pilin			Gonococcus recombinant pilin+Stimulon ^TM QS-21
The gonococcus bacterial strain	Meningococcus reorganization I class pilin			Gonococcus recombinant pilin+Stimulon ^TM QS-21		Stimulon ^TM QS-21	AlPO ₄	Do not have
I756	301,969	122,714	78,111	46,424		Stimulon ^TM QS-21	AlPO ₄	Do not have
I756	301,969	122,714	78,111	46,424	FA1090	322,311	262,422	170,842	108,094

Embodiment 23

With the chimeric I class of meningococcus recombinant pilin antiserum(antisera) passive protection body opposing meningococcus microbemia

The initial young rat model of describing of Saukkonen and Leinonen (34) is a kind of feasibility animal model of estimating vaccine protection body energy meningococcemia microbemia ability.Tested the chimeric I class of meningococcemia recombinant pilin guinea pig antiserum (with Stimulon ^TMQS-21 is an adjuvant) protect young rat to resist the ability of microbemia (causing) by the meningococcus bacterial strain of expressing pilin.At the 0th day, the anti-chimeric I class recombinant pilin guinea pig antiserum (the 6th week) of Sprague-Dawley children rat (4-5 age in days) passive immunization (peritoneal injection) 0.1mL (to be diluted in PBS in 1: 5,1: 10 or 1: 20).Control group is accepted 0.1mL normal guinea pig serum (the 0th week) (to be diluted in PBS at 1: 5) injection.After 24 hours, with about 5 * 10 ⁵The 0.1mL of colony-forming unit (cfu) mao meningococcus (bacterial strain H355) that carries disease germs is attacked (peritoneal injection) animal.Attack after 3 hours, put to death animal, get the dilution of equal portions painstaking effort, place on the GC agar plate.Then at 37 ℃ of 5%CO ₂The middle cultivation dull and stereotyped 18-24 hour.Enumeration of bacterial colonies is determined bacteremic level then.Compared with a kind of variance t check analysis method and to have accepted immune serum (the 6th week) and organize and accept normal serum (the 0th week) control group.As shown in following table 14, compare with the animal that inoculates with the normal guinea pig seroimmunity, use the animal of the guinea pig antiserum passive immunization of the chimeric I class of specificity meningococcemia recombinant pilin, show the microbemia level and be the minimizing of logarithm level.This difference has statistical significance, p＜0.05.

Table 14

The guinea pig antiserum of the chimeric I class of meningococcemia recombinant pilin is with mao meningococcus (bacterium that carries disease germs

Strain H355) prevents bacteremic ability in the young rat of Gong Jiing ^*

Blood sampling	Extent of dilution	Cfu mean value ± std
Blood sampling	Extent of dilution	Cfu mean value ± std	The 0th week	1∶5	4.87±0.18
The 6th week	1∶5	3.55±0.48 ^**	The 0th week	1∶5	4.87±0.18
The 6th week	1∶5	3.55±0.48 ^**	The 6th week	1∶10	3.63±0.36 ^**
The 6th week	1∶20	3.98±0.75 ^**	The 6th week	1∶10	3.63±0.36 ^**

^*As described in Table 7, from the chimeric I class of the meningococcemia recombinant pilin antiserum(antisera) of cavy acquisition.

^**p＜0.05。

Cfu ± std=colony-forming unit ± standard deviation

Embodiment 24

Induce the mucosal immune response of the chimeric I class of meningococcemia recombinant pilin

In the 0th, 1 and 2 weeks, give the chimeric I class of immunization meningococcus recombinant pilin salt solution (5 μ g/10 μ L) in the mouse nose, wherein contain or do not contain the cross reaction mutant form Toxins,exo-, cholera (CT-CRM, E29H).As shown in table 15 below, when animal is used the recombinant pilin immunization of no adjuvant, in antigen ELISA, measure significant immunne response.The adding Toxins,exo-, cholera has improved this and has replied.

Table 15

The reorganization meningococcus of the chimeric I class of meningococcemia recombinant pilin mixing mouse resisting anteserum and purifying

Chimeric I class recombinant pilin bonded terminal point titre ^*

	Recombinant pilin (no adjuvant)	Recombinant pilin adds CT-CRM
	Recombinant pilin (no adjuvant)	Recombinant pilin adds CT-CRM	Serum IgG IgA	6,168 490	1,181,871 3,940
The segmental bronchus washing lotion ^** IgG IgA	＜10 ＜10	580 19	Serum IgG IgA	6,168 490	1,181,871 3,940
The segmental bronchus washing lotion ^** IgG IgA	＜10 ＜10	580 19	The nasal cavity washing lotion ^** IgG IgA	＜10 12	98 236
Vaginal douche ^** IgG IgA	174 15	70 687	The nasal cavity washing lotion ^** IgG IgA	＜10 12	98 236

^*Analyzed the pooled serum in the 4th week.The IgG of the 0th all pooled serums and IgA terminal point titre＜50.

^*Followingly carry out lavation:

Segmental bronchus: with 1mL RPMI 1640 lavation lungs 5 times, in sample, add 50 μ L foetal calf serums (FBS) then, centrifugal (2,000 * g * 5 minute) clarification ,-20 ℃ of preservations.

Nasal cavity: with 0.5mL RPMI 1640 lavation nostrils once, in sample, add 20 μ L FBS, ℃ preservations then-20.

Vagina: use 0.075mL RPMI 1640 lavation vaginas 5 times, in sample, add 10 μ L FBS ,-20 ℃ of preservations then.

Embodiment 25

Initiatively protect with the chimeric I class of meningococcus recombinant pilin antiserum(antisera) and to resist meningococcal settling down

The first step of the scorching coccus disease of human body endomeninx is that bacterium is settled down in nasopharynx.In this course, it is believed that pili plays a major role in mediating bacterium and epithelial cell tentatively contacts.Many investigators have described the process of bacterial colonisation in the new born animal nasopharynx, but nobody studies (35) with its model of rendeing a service as the test meningococcus vaccine.In order to estimate the present invention, developed with ripe outbreeding mouse as the model of settling down in the meningococcus nose.In the 0th, 4 and 8 weeks, to the chimeric I class of the subcutaneous immunization meningococcus of Swiss-Webster mouse recombinant pilin (with MPL ^TMBe adjuvant).In the 10th week, animal intraperitoneal injection 2mg dextran iron (Sigma), and with 10 μ L about 1 * 10 ⁷Attack in mao meningococcus that carries disease germs (the containing 40 μ g dextran iron equally) nose in logarithmic growth mid-term of cfu.Attacked the back the 1st day, half animal is accepted intraperitoneal injection 2mg dextran iron again.Attacked the back the 1st and 2 day, and, measured amount of viable bacteria in the nose by the nose tissue homogenate is inoculated on the GC agar plate that contains the selective antibiotic element.The results are shown in table 16.

Table 16

The viable count (cfu) that reclaims in the mouse nose homogenate of attacking with the meningococcus bacterial strain H355 of the hair that carries disease germs ^*

Antigen (dosage μ g)	The cfu/ nose
	The cfu/ nose		The 1st day ^**	The 2nd day ^**
	The full cell of H355 (25)	1,165	The 1st day ^**	The 2nd day ^**	67
I class recombinant pilin (10)	The full cell of H355 (25)	1,165	6,866	63	67
I class recombinant pilin (10)	Salt solution	17,943	6,866	63	3,406

^*All vaccines are with 100 μ g MPL ^TM/ agent preparation.Form by 5 mouse for every group.

^*Fate after attacking in the nose.

Embodiment 26

The immunne response of the chimeric recombinant pilin of Western engram analysis meningococcemia II class

Adopt as embodiment 11 described flow processs, with the chimeric recombinant pilin immunization of the meningococcus II class cavy of purifying.At first analyzed the antiserum(antisera) (not having display data) that produces with the cavy of the chimeric recombinant pilin immunization of meningococcus II class with the Western blotting.These traces show that the chimeric recombinant pilin antiserum(antisera) of meningococcemia II class has been discerned a band corresponding to pilin in the full cell lysate of mao meningococcus cell that carries disease germs (expressing II class pilin (FAM18) or I class pilin (H355)).On the contrary, the anti-antiserum(antisera) that contains the intestinal bacteria extract of pTrcHis carrier does not react with any pilin band in the Western trace.

Embodiment 27

The chimeric II class of meningococcemia recombinant pilin combines with mao meningococcus cell that carries disease germs

The antiserum(antisera) of the anti-chimeric II class of partially purified meningococcus recombinant pilin shows the meningococcus cell that can be incorporated into homologous strain FAM18, titre＞36,450 (titre in the 0th week is 473).

Reference

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Sequence table

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<212>DNA

＜213〉Diplococcus gonorrhoeae

<400>10

ggctctagat tagaaaaact catcgagc 28

<210>11

<211>36

<212>DNA

＜213〉I class Neisseria meningitidis

<400>11

ccccgcgcca tggacaccct tcaaaaaggt tttacc 36

<210>12

<211>39

<212>DNA

＜213〉I class Neisseria meningitidis

<400>12

gggcctggat ccgagtggcc gtggaaaatc acttaccgc 39

<210>13

<211>31

<212>DNA

＜213〉I class Neisseria meningitidis

<400>13

ccggcgcgtc tctcacggcg aatggcccgg c

31

<210>14

<211>39

<212>DNA

＜213〉I class Neisseria meningitidis

<400>14

gggcctggat ccgagtggcc gtggaaaatc acttaccgc 39

<210>15

<211>25

<212>DNA

＜213〉Diplococcus gonorrhoeae

<400>15

gcataattcg tgtcgctcaa ggcgc 25

<210>16

<211>34

<212>DNA

＜213〉Diplococcus gonorrhoeae

<400>16

gccgcgcgtc tcccgtgatt caggtaatac tcgg 34

<210>17

<211>25

<212>DNA

＜213〉I class Neisseria meningitidis

<400>17

gcataattcg tgtcgctcaa ggcgc 25

<210>18

<211>39

<212>DNA

＜213〉I class Neisseria meningitidis

<400>18

gggcctggat ccgagtggcc gtggaaaatc acttaccgc 39

<210>19

<211>37

<212>DNA

＜213〉II class Neisseria meningitidis

<400>19

gcggccgcca tggaagcaat ccaaaaaggt ttcaccc 37

<210>20

<211>33

<212>DNA

＜213〉II class Neisseria meningitidis

<400>20

gccgcgcgtc tccgaaccgg agttttgttt gcc 33

<210>21

<211>33

<212>DNA

＜213〉Diplococcus gonorrhoeae

<400>21

ccgggccgtc tcggttcggt aaaatggttc tgc 33

<210>22

<211>30

<212>DNA

＜213〉Diplococcus gonorrhoeae

<400>22

gggcctggat ccgtgggaaa tcacttaccg 30

<210>23

<211>37

<212>DNA

＜213〉II class Neisseria meningitidis

<400>23

gcggccggat ccggtcattg tccttatttg gtgcggc 37

<210>24

<211>22

<212>PRT

＜213〉Diplococcus gonorrhoeae

<400>24

Glu Ala Ile Leu Leu Ala Glu Gly Gln Lys Ser Ala Val Thr Glu Tyr

1 5 10 15

Tyr Leu Asn His Gly Lys

20

Claims

1. a vaccine composition is characterized in that, the recombinant expressed Nai Seshi that it comprises separation and purification belongs to pilin, and described pilin is made up of following three zones: (a) the aminoterminal zone of high conservative; (b) region intermediate, this district shows limited amount sequence variations and (c) proteic third part carboxyl terminal, wherein said vaccine composition can cause protective immune response in the human host.

2. vaccine composition as claimed in claim 1 is characterized in that described pilin is from Diplococcus gonorrhoeae.

3. vaccine composition as claimed in claim 1 is characterized in that described pilin is from Neisseria meningitidis.

4. vaccine composition as claimed in claim 3 is characterized in that, described pilin is a Neisseria meningitidis I class pilin.

5. vaccine composition as claimed in claim 3 is characterized in that, described pilin is a Neisseria meningitidis II class pilin.

6. vaccine composition as claimed in claim 1, it is characterized in that, described pilin is the chimeric recombinant pilin of Diplococcus gonorrhoeae and I class Neisseria meningitidis, described pilin has the aminoacid sequence of SEQ ID NO:2 amino acid/11-167 before processing, or has the aminoacid sequence of SEQID NO:2 amino acid 8-167 after being processed into maturation protein.

7. vaccine composition as claimed in claim 1 is characterized in that described vaccine composition also comprises adjuvant, diluent or carrier.

8. vaccine composition as claimed in claim 7 is characterized in that, described adjuvant is selected from: aluminium hydroxide, aluminum phosphate, Stimulon ^TMQS-21,3-O-deacylated tRNA monophosphoryl lipid A, IL-12 and wild-type or mutant Toxins,exo-, cholera.

9. the purposes of the described vaccine composition of claim 2 is characterized in that, this vaccine composition is used to prepare the medicine of anti-Diplococcus gonorrhoeae.

10. the purposes of the described vaccine composition of claim 3 is characterized in that, this vaccine composition is used to prepare the medicine of anti-Diplococcus gonorrhoeae.

11. the purposes of the described vaccine composition of claim 6 is characterized in that, this vaccine composition is used to prepare the medicine of anti-Diplococcus gonorrhoeae.

12. the purposes of the described vaccine composition of claim 3 is characterized in that, this vaccine composition is used to prepare the medicine of anti-Neisseria meningitidis.

13. the purposes of the described vaccine composition of claim 2 is characterized in that, this vaccine composition is used to prepare the medicine of anti-Neisseria meningitidis.

14. the purposes of the described vaccine composition of claim 6 is characterized in that, this vaccine composition is used to prepare the medicine of anti-Neisseria meningitidis.

15. method for preparing the vaccine composition of claim 1, it is characterized in that, the method comprising the steps of: adding dosage is enough to make described vaccine composition to cause protective immune response in the human host, the recombinant expressed Nai Seshi of separation and purification belongs to pilin, and described pilin is made up of following three zones: (a) the aminoterminal zone of highly protecting; (b) region intermediate, this district shows limited amount sequence variations and (c) proteic third part carboxyl terminal.

16. the dna sequence dna of separation and purifying, it is characterized in that, described dna sequence dna comprises the dna sequence dna of coding Diplococcus gonorrhoeae and the chimeric recombinant pilin of I class Neisseria meningitidis, described pilin has the aminoacid sequence of SEQ ID NO:2 amino acid/11-167 before processing, or has the aminoacid sequence of SEQ ID NO:2 amino acid 8-167 after being processed into maturation protein.

17. the dna sequence dna of separation and purification as claimed in claim 16 is characterized in that, described dna sequence dna has the nucleotide sequence of SEQ ID NO:1 Nucleotide 1-501 or 22-501.

18. plasmid, it is characterized in that, described plasmid includes the dna sequence dna of a kind of separation and purifying, this sequence comprises the dna sequence dna of the chimeric recombinant pilin of coding Diplococcus gonorrhoeae and I class Neisseria meningitidis, described pilin has the aminoacid sequence of SEQ ID NO:2 amino acid/11-167 before processing, or has the aminoacid sequence of SEQ ID NO:2 amino acid 8-167 after being processed into maturation protein.

19. plasmid as claimed in claim 18 is characterized in that, described plasmid comprises the dna sequence dna with SEQ ID NO:1 Nucleotide 1-501 or 22-501 nucleotide sequence.

20. plasmid as claimed in claim 19 is characterized in that, described plasmid is named as ATCC98637.

21. one kind with the described plasmid transformed host cells of claim 18.

22. host cell as claimed in claim 21 is characterized in that, described host cell is the colon bacillus bacterial strain.

23. host cell as claimed in claim 22 is characterized in that, described plasmid is named as ATCC98637.

24. method that produces the chimeric recombinant pilin of Diplococcus gonorrhoeae and I class Neisseria meningitidis, it is characterized in that, this method comprises: transform or transfection host cell with the described plasmid of claim 19, and cultivate this host cell under the condition that allows the described chimeric recombinant pilin of host cell expression.

25. a separation and the Diplococcus gonorrhoeae of purifying and the chimeric recombinant pilin of I class Neisseria meningitidis, it is characterized in that, described pilin has the aminoacid sequence of SEQ ID NO:2 amino acid/11-167 before processing, or has the aminoacid sequence of SEQ ID NO:2 amino acid 8-167 after being processed into maturation protein.

26. vaccine composition as claimed in claim 1, it is characterized in that, described pilin is the chimeric recombinant pilin of Diplococcus gonorrhoeae and II class Neisseria meningitidis, described pilin has the aminoacid sequence of SEQ ID NO:4 amino acid/11-170 before processing, or has the aminoacid sequence of SEQ ID NO:4 amino acid 8-170 after being processed into maturation protein.

27. the purposes of the described vaccine composition of claim 26 is characterized in that, is used to prepare the medicine of anti-Diplococcus gonorrhoeae.

28. the purposes of the described vaccine composition of claim 26 is characterized in that, is used to prepare the medicine of anti-Neisseria meningitidis.

29. the dna sequence dna of separation and purifying, it is characterized in that, described dna sequence dna comprises the dna sequence dna of coding Diplococcus gonorrhoeae and the chimeric recombinant pilin of II class Neisseria meningitidis, described pilin has the aminoacid sequence of SEQ ID NO:4 amino acid/11-170 before processing, or has the aminoacid sequence of SEQ ID NO:4 amino acid 8-170 after being processed into maturation protein.

30. the dna sequence dna of separation as claimed in claim 29 and purifying is characterized in that, described dna sequence dna has the dna sequence dna of SEQ ID NO:3 Nucleotide 1-510 or 22-510 nucleotide sequence.

31. plasmid, it is characterized in that, described plasmid comprises the dna sequence dna of a kind of separation and purifying, this sequence comprises the dna sequence dna of the chimeric recombinant pilin of coding Diplococcus gonorrhoeae and II class Neisseria meningitidis, described pilin has the aminoacid sequence of SEQ ID NO:4 amino acid/11-170 before processing, or has the aminoacid sequence of SEQ ID NO:4 amino acid 8-170 after being processed into maturation protein.

32. plasmid as claimed in claim 31 is characterized in that, described plasmid comprises the dna sequence dna with SEQ ID NO:3 Nucleotide 1-510 or 22-510 nucleotide sequence.

33. plasmid as claimed in claim 31 is characterized in that, described plasmid is named as ATCC207199.

34. one kind with the described plasmid transformed host cells of claim 31.

35. host cell as claimed in claim 34 is characterized in that, described host cell is the colon bacillus bacterial strain.

36. host cell as claimed in claim 35 is characterized in that, described plasmid is named as ATCC207199.

37. method that produces Diplococcus gonorrhoeae and the chimeric recombinant pilin of II class Neisseria meningitidis, it is characterized in that, this method comprises: transform or transfection host cell with the described plasmid of claim 31, and cultivate this host cell under the condition that allows the described chimeric recombinant pilin of host cell expression.

38. the Diplococcus gonorrhoeae and the chimeric recombinant pilin of II class Neisseria meningitidis of separation and purifying, described pilin has the aminoacid sequence of SEQ ID NO:4 amino acid/11-170 before processing, or has the aminoacid sequence of SEQ ID NO:4 amino acid 8-170 after being processed into maturation protein.