CN1067678A - The B oligomer of pertussis toxin of reorganization - Google Patents

Abstract

Can in antipertusis vaccine, be used as the B oligomer of pertussis toxin sample polymer protein of the genetically engineered transformation of non-reactionogenicity component.This protein is by the composition of cistron separately of expressing subunit S2, S3, S4 and S5 respectively in heterologous host, reclaims the subunit polypeptide of gained, and makes it to produce to be assembled into the polymer synthetics in external combination.

Description

The B oligomer of pertussis toxin of reorganization

The present invention relates to the recombinant expressed of B oligomer of pertussis toxin subunit, and subunit can be used in non-reactionogenicity polymer component in the vaccine in external combination with formation, belong to the protective immune response that causes infection to cause anti-bordetella.

Requirement to the untoward reaction that reduces pertussis vaccine impels people to study to understand the immunogenic components of pathogenic agent Whooping cough bordetella better.An important component of new acellular vaccine is Toxins, pertussis (PT), and it is for disturbing the different six aggressiveness ADP-ribosyltransferases (1-3) of hormone signal transduction in the mammalian cell.Be present in this kind of enzyme in the toxin S1 subunit (4) and urge activity to cause toxicity, and be commonly considered as causing the root of untoward reaction the pertussis vaccine recipient.Therefore, in producing toxin base pertussis vaccine, major concern be the enzymatic activity of removing the S1 subunit.At present, generally be to handle the vaccine material with toxoid agent such as heat or formaldehyde and glutaraldehyde, make S to reach ₁The inactivation purpose.The shortcoming of these physics method for deactivating is that strictly speaking, their essence has reduced the protective immunity originality of toxin; The toxin dependency enzymic activity (5) that molecule then can after depositing, occur even so.

Developed in intestinal bacteria and produced each Toxins, pertussis subunit (6), and identified key zone relevant on the S1 subunit and form the method that the advantage protective antigen determines base (7) with enzymatic activity with recombinant technology.By site-specific mutagenesis, can eliminate the enzymatic activity of S1 subunit, obtain and form protective antigen decision basic (8) or make this subunit combine the conformation that adapts with assembling or holotoxin sample polymer (9) with the B oligomer with infringement S1 subunit.

It is believed that the B oligomer has the cell receptor identified region, it is the transmission platform (1.10-13) of S1 subunit; It can be used as does not have the S1 subunit, and by subunit S2, S3, S4 and S5, to be respectively 1: 1: 2: the inferior aggressiveness macromole that 1 approximate ratio is formed separates and obtains (1).Prove that recently not having the ADP-ribosyltransferase active natural B oligomer relevant with Toxins, pertussis toxicity originally itself promptly is enough to induce protective immunity reaction (14-16); In addition, in the recombinant protein research of cooperating to carry out with U.S. food and Drusilla doctor L.Burns of Drug Administration and Juan doctor L.Arciniega, show that as if the S1 subunit to the not obviously contribution of aversion response of B oligomer.Therefore, the B oligomer obviously has the possibility as acellular pertussis vaccine composition.Yet, employed natural B oligomer is isolating from the holotoxin molecule in these researchs, and do not have S1 subunit (＞95%) mostly, so but the B oligomer that obtains by this way has the S1 related activity of detection limit, and this activity plays great role to its reactionogenicity.

Fig. 1 shows the recombinate SDS-PAGE collection of illustrative plates of B oligomer sample polymer (hereinafter to be referred as the B oligomer) of the present invention.The B oligomer is to form in assembled in vitro by described subunit S2, S3, S4 and the S5 that is produced by the recombinant chou intestinal bacteria hereinafter, and on Pp63 glycophosphoproteins-Sepharose post (17) of affinitive layer purification.Used 4M MgCl with trichoroacetic acid(TCA) (TCA) precipitation ₂The sample of wash-out from the chromatography column, and carry out SDS-PAGE(19).Use Coomassie blue stain then.Swimming lane 1) is natural PT(commercial grade); Swimming lane 2) is natural B oligomer (commercial grade); Swimming lane 3) is reorganization B oligomer.

Fig. 2 is that diagram shows B oligomer preparation, comprises the short cell fission activity of reorganization B oligomer preparation of the present invention.In the RPMI1640 substratum with the pointed B oligomer preparation of twice serial dilution.In B oligomer preparation, add mouse spleen lymphocyte then, reach B oligomer concentration as indicating on the abscissa.With ordinary method detect [ ³H] thymidine mixes (19).Because reorganization B oligomer preparation is stored in the high concentration urea (2M), and because of its initial protein concentration is quite low, so contain the residual urea that can cause necrocytosis in a large number completely in the low dilution reorganization B oligomer preparation.Each preparation is: natural B oligomer (0); Show the natural B oligomer (b) that contains 0.125M urea of maximum concentration; And the reorganization B oligomer that contains 0.125M urea demonstration maximum concentration.

Fig. 3 illustrates with B oligomer preparation immune mouse the leukocytosis of PT is promoted active influence.5 mouse are respectively through intraperitoneal injection (i.p.) reorganization B of the present invention oligomer preparation (rB) or natural B oligomer preparation (B), being added in cumulative volume is that dosage is respectively 8 μ g(rBM/BH in the PBS-gel that contains adsorbed onto alum adjuvant of 0.5ml) 1 μ g(rBM/BM) or 0.1 μ g(rBL/BL) (seeing Table 1).One group of 5 mouse (indicating " PT ") are immune in contrast with the PBS-gel.After one month, each is organized mouse and all uses 500ngPT(i.p.) attack and (but to be denoted as " PBS ^*" group except, this group is attacked with PBS); Survey white blood cell count(WBC) after 4 days.The result adds a standard deviation with the mean value of white blood cell count(WBC) and represents.

The invention provides the B oligomer sample polymer protein (reorganization B oligomer) of the genetically engineered transformation of the Toxins, pertussis that is derived from recombinant material, with and preparation method thereof.Briefly; produce the cistron composition (" gene ") and the derivative thereof of S2, S3, S4 and S5 subunit and these subunit analogs of toxin through genetic manipulation (as site-specific mutagenesis); and in allos (" the external ") host that difference transforms, express respectively and gather in the crops it; then in the external polymer synthetics that is combined into similar natural B oligomer; this synthetics has the ability that causes mitosis reaction in white corpuscle, and the more important thing is the immunoprotection reaction that can induce anti-Toxins, pertussis.

With reorganization of the present invention B oligomer or from the PT that Bordetella pertussis produces isolating natural B oligomer immune mouse, be created in show in the elisa assay can with PT reaction and energy in and PT to the antibody of the cytopathic effect of Chinese hamster ovum (CHO) cell cultivated; The antibody titers all similar of bringing out by each preparation.In addition, can resist the leukocytosis promotion activity of PT with natural or reorganization B oligomer mice immunized.These digital proofs are to participate in the suitable candidate's composition that constitutes the acellular pertussis vaccine at external B oligomer of pertussis toxin of the present invention by indivedual reorganization subunit assemblings.

In recombination bacillus coli, produced each subunit (6) by former described method.In this recombinant chou production system, replace its intrinsic signal peptide sequence with synthetic chairman of committee's subunit polypeptide with initial methionine residue; Except that subunit S4, allos N-terminal methionine residue is in fact all cut away from each recombinant protein matter by endogenous methinyl aminopeptidase.Therefore the S4 subunit is a kind of " methinyl maturation " polypeptide, that is to say that it is the analogue that carries the natural ripe S4 subunit of methionine residue at its N-terminal.Ferment respectively with synthetic each recombinant chou protein subunit matter in intestinal bacteria recombinant chou separately.From fermented liquid, reclaim cell mass, place-60 ℃ to-80 ℃ to preserve down then.In about 4 ℃, in the presence of 1mM dithiothreitol (DTT) (DTT), making cell mass is about 10 by pressure twice ⁴The French of psig squeezes device, with smudge cells.Under these fermentation conditions, recombinant chou protein subunit matter is as the insoluble inclusion body synthetic in the intestinal bacteria.From cellular lysate, reclaim these inclusion bodies, and wash at least twice with differential centrifugation; Be at 1% Septochol (DOC) and 25mM Tris-HCl(pH7.9 for the first time) in, be at 4M urea and 25mM Tris-HCl(pH7.5 for the second time) in.

Various inclusion body preparations are weighed, and according to the relative percentage example of they and protein subunit matter, promptly their approximate molar ratio in the natural B oligomer are mixed it with stoichiometric quantity (S2: S3: S4: S5 were respectively 1: 1: 2: 1).Be added in 25mM Tris-Hcl(pH8.5) in 6M hydrochloric acid Guanidinium (GuHCl) and 10mM DTT solution, with dissolving inclusion body mixture; At room temperature mixture is stirred gently then and spend the night.Centrifugal (use the JA20 rotor, with 18,000rpm under 2 ℃ of room temperatures centrifugal 30 minutes) to remove insoluble material.Reclaim the solubility supernatant, and usefulness does not contain the above-mentioned GuHCl-Tris buffer solution elution of DTT to remove too much DTT in the GH25 chromatography column.Be recovered in or the protein peak part of the void volume place wash-out of approximate GH25 post.Can use the 6MGuHCl elution samples in any time after this step.

Add Cu ₂SO ₄To final concentration be 50 μ M; Under room temperature and atmospheric environment, sample slowly stirred to tremble and spend the night.After this re-oxidation step, make sample to contain twice (the using 5 liters) of 100mM potassium phosphate buffer (pH7.4) dialysis of 2M urea at every turn.The tubulature of/dialysis bag should have 3,500 daltonian molecular weight cut-offs.Form a kind of insoluble precipitate between dialysis period, its can through centrifugal (the JA14 rotor, 13,000rpm, under the room temperature centrifugal 20 minutes) remove.Collecting supernatant preserves down in 2-8 ℃.Then with sample to phosphate buffered saline (PBS) (PBS) dialysed overnight under room temperature; Dialysate volumes should be no more than 10 times of sample volume, and should change liquid once at least.In addition, the dialysis bag tubulature should have 3,500 daltonian molecular weight cut-offs.

Prepare Pp63 glycophosphoproteins-Sepharose affinity column with commercially available Pp63 glycophosphoproteins Sepharose 4B, with Spiro method (available from Gibco) purifying, and covalently bound under disclosed condition.The sample of dialysis is gone up Pp63 glycophosphoproteins-Sepharose chromatography column at twice under 2 ℃ of room temperatures; Also can utilize a prose style free from parallelism (no post) chromatography, washing and elution process.(0.05M Tris-HCl pH7.5) washes the affinity resin of going up sample with the buffer A that contains 1M NaCl with PBS in succession.With containing 4M MgCl ₂The buffer A wash-out be attached to sample on the resin under these conditions.

All things considered can separate the subunit of respectively recombinating as insoluble inclusion body behind dissolution of bacteria.Each inclusion body preparation is dissolved among the 6M hydrochloric acid Guanidinium (GuHCl) that is added with 1mM dithiothreitol (DTT) (DTT), and (be S2: S3: S4: S5 was respectively 1: 1: 2: 1) in conjunction with each subunit with reference to the approximate molar ratio example of the estimation in the natural B oligomer.Use based on the exclusion chromatography of molecular size and remove reductive agent, at 50 μ M Cu ₂SO ₄Exist to stir to tremble in atmospheric environment down to make it to reoxidize, and 2M urea is carried out, equilibrium dialysis is beneficial to the spontaneous combination of each subunit.By its ability [a kind of cell of toxin hits target drone system (20)], be purified to homogeneous degree (purity of each subunit is estimated it in according to polymer) (Fig. 1) by on Pp63 glycophosphoproteins-Sepharose post, carrying out the affinity chromatography B oligomer of will recombinating in conjunction with complex carbohydrates; In our experience, indivedual subunits almost can not combine specifically with the Pp63 glycophosphoproteins resin, and this shows most of at MgCl ₂The material of middle wash-out is the polymer form.The check eluate finds that its agglutination reaction to goose hematid is positive.Carry out Western with the polyclone antitoxic serum and inhale seal analysis, and the ELISA that improves test (use flat board and anti-S2, S3, S4 and the S5 monoclonal antibody of Pp63 glycophosphoproteins bag quilt, and compare with anti-S1 monoclonal antibody) further confirms the existence of each immunoreactivity B subunit polypeptide.Though subunit S2 and S3 and ratio (painted SDS-polyacrylamide being detected with the integration scanning optical densitometric method) and natural B oligomer are slightly different, this may be interpreted as and has too much S2 subunit (it is described to see above) or the S3-S4 dimer of co-elute from the affine resin of Pp63 glycophosphoproteins-Sepharose.But obviously can experimental results show that isolating polymer contains five yuan of B oligomer (Fig. 2) of significant quantity by mitogenesis; And B oligomer and some indivedual subunit and the dimer that contains it can make erythrocyte aggregation, and have only complete B oligomer that mitogenic activity is arranged.It should be noted that no matter the relative proportion of indivedual protein subunits (S2, S3, S4 and S5) how, reorganization B oligomer of the present invention is different from the B oligomer of natural form eventually, because of wherein it has had methinyl one sophisticated S4 subunit.

Use a kind of standard method of estimating the usefulness of pertussis vaccine then, detect reorganization B oligomer preparation in the immunoreactive ability of laboratory animal moderate stimulation toxin neutrality.This test method is to be finished by doctor D.L.Burns of FDA and doctor J.L.Arciniega.At first inoculate mouse through intraperitoneal injection with the natural of definite dosage or reorganization B oligomer.In two weeks of immunity back, the mouse bloodletting to be detecting the concentration of t antibody with the ELISA method, and then attacks it with lethal quantity PT; Observe the death toll that animal causes because of toxin, and in the back leukocytosis of estimating the toxin mediation of all animals in 4 days of attack.As table 1 the result proved, the mouse that each immunizing agent is reacted all has in tangible toxinicide and the toxin and concentration, wherein the latter is according to the Chinese hamster ovary celI bunch collection of PT mediation reduced to act on mensuration; Observed concentration was similar when in addition, reorganization B oligomer inductive NAT was made immunogen to use natural B oligomer.

Table 1

Immunogen ^aAmong the B oligomer PT and humidity ^c

The natural B oligomer

1 ° of immunity 4,838 2,478 226

2 ° of immunity 223,664 41,085 1,810

Reorganization B oligomer

1 ° of immunity 12,068 1,933 113

2 ° of immunity 144,813 79,834 2,506

A, under room temperature, the antigen with phosphate buffered saline (PBS) (PBS-gel) dilution that contains 0.02% gelatin was adsorbed onto on the aluminium hydroxide that is equivalent to gross protein (antigen adds gelatin) amount through 1 hour, then under 4 ℃ to the PBS dialysis to remove urea and salt.1 group 5 BALB/C female mices (14-16g) are respectively through intraperitoneal injection (i.p.) antigen (6 μ g).After 29 days, bloodletting also prepares serum.Second day, once more to mouse carry out immunity (2 μ g antigen/mouse, i.p.).After 14 days to the mouse bloodletting and prepare serum.

B, each numerical value are that the routine number as the antilogarithm of the x intercept that obtains from the extrapolation of the linear portion of each curve calculates, and wherein said curve is log ₁₀Mapping obtains serum dilution to 405nm place light absorption ratio value; The result that on behalf of the combining anteserum by 5 mouse, each numerical value record.Injection PBS-gel+Al(OH) ₃The serum of mouse do not show anti-B oligomer or the PT titre that can measure.

C, by stating the ability that method (25) detects in the serum and Toxins, pertussis is induced Chinese hamster ovary celI is trooped.Numerical value is to represent in the holotoxin and the Chinese hamster ovary celI dilution expression reciprocal of active maximum serum (serum of 5 mouse of merging) of trooping.Injection PBS-gel+Al(OH) ₃The serum of mouse do not show the neutralizing antibody concentration that can measure.

The minimum dilution of test is 1: 20.

The more important thing is that in natural B oligomer or the animal with the immunity of reorganization B oligomer, the provide protection level that the toxicity of the antagonism PT that is given is made effect does not have notable difference (Fig. 3).Indivedual subunits (6,22) of B oligomer and indivedual S2-S3 or S3-S4 dimer (23) all can not cause the immanoprotection action of the leukocytosis accelerating effect of antagonism PT.Though lowest dose level (the 0.1 μ g) effectiveness of recombined material may be lower than the effectiveness of isodose natural B oligomer slightly, this species diversity is likely because due to painted SDS-polyacrylamide gel being integrated the accuracy difference that spectrodensitometry exists when determining the protein concn of reorganization B oligomer.

The result of Ti Chuing has proved the feasibility that is produced compound different glycoprotein polyprotein precursor matter by recombinant DNA deutero-subunit herein.For PT, might produce the inferior lps molecule form of high immunogenicity now.Though can be by in its coding cistron structure, causing sudden change.Make the S1 subunit in Bordetella pertussis, produce the B oligomer in conjunction with the ability inactivation of B oligomer, even but also considerably less (24) are arranged by the B oligomer of this microorganisms.Also can be from natural toxin purifying B oligomer, but this is a very big method of cost workload, and still can cause the detectable pollution (9,19,25) that contains due to the S1 holotoxin.Reorganization B oligomer of the present invention is not only owing to disappearance S1 subunit does not have its inherent toxicity, face and because of in manufacturing processed, having removed Pathogenic organisms, so also eliminated the possibility (26 of polluting other Bordetella pertussis toxic components, 27), and the latter also may be the reason of the untoward reaction of pertussis vaccine.

Can be according to a conventional method the B oligomer sample polymer of genetically engineered transformation of the present invention be made into vaccine.Toxin " in the heredity " by the situation of inactivation under, Toxins, pertussis for example of the present invention, generally needn't use other inactivation step (as chemical treatment or thermal treatment), because these materials produce in the non-virulent organism, and originally just do not have general flat think can cause S1 related enzyme activity to the untoward reaction of treated (or do not have strict inactivation) the Whooping cough holotoxin vaccine in whole cell pertussis vaccine and end.However, still be necessary to control the purity of recombinant product, particularly control endotoxin content.In general, the reorganization B subunit of describing among the present invention should be purified to 〉=90% uniformity as the vaccine inoculation antigen that potential using value is arranged.If any, should evaluate the character and the estimation amount of pollutent, meet the standard of each administration with the degree that guarantees contaminated with endotoxins.

Carry in order to carry out the parenteral approach, generally the vaccine material should be adsorbed onto on the aluminium adjuvant.These available at least two kinds of methods are finished: with the alum coprecipitation of preliminary working and with aluminium salt coprecipitation.Throw out with absorption is suspended in the vehicle then, be generally every dose of 5-100 μ g to obtain single agent concentration, and the aluminium hydroxide amount is no more than the vaccine antigen of every dose of 1.5mg; Every dose of volume is generally in the 0.1-1.0ml scope.Suspension vehicle normally buffered soln (as phosphate buffered saline (PBS), pH7.0), and can contain stablizer (as glycerine) and sanitas to prevent microbial contamination and to prolong effective shelf lives.In order to stimulate secretion property mucosal immunoreaction, should be able to make antigen be transported to the Lymphoid tissue of sticking to mould.In this respect, the vaccine material can be made enteric coated capsule or other deliver carrier, to prevent that arriving road intestines Lymphoid tissue (as peyer's patches) at it is degraded before.In enteron aisle, cause the lamina propria that secondary immune response can fully be delivered to the wrinkling organ of other mucous membranes, particularly be delivered to respiratory tract with the prevention Whooping cough.In addition, vaccine product can be added in suitable vehicle and/or transport and be made into aerosol or the liquid that is suitable for through respiratory tract, mouth or nasal administration in the carrier (as liposome), vaccine can directly stimulate secondary immune response or transmit (as mentioned above) lamina propria to respiratory tract from mouth or nose Lymphoid tissue in segmental bronchus like this.

Though for example understand the present invention by particular instance, should be clear and definite be that mode of the present invention is done some other change and change is possible implementing.The present invention will comprise all these changes and the change that belongs in the scope of the invention that claim limited that awaits the reply.

Reference list

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Claims (23)

1, comprise the B oligomer of pertussis toxin sample polymer protein of subunit S2, S3, S4 and S5, wherein S4 is the methinyl mature polypeptide, or this polymeric analogue or derivative.

2, according to the B oligomer of pertussis toxin sample polymer protein of claim 1, wherein stoichiometrical molar ratio of subunit S2, S3, S4 and S5 was respectively about 1: 1: 2: 1.

3, antipertusis vaccine, it comprises the claim 1 of significant quantity or 2 B oligomer of pertussis toxin sample polymer protein.

4, the method for producing B oligomer of pertussis toxin sample polymer protein by the reorganization subunit; this method comprises the cistron composition of expressing subunit S2, S3, S4 and S5 respectively in the heterogenous expression host; or its any locus specificity codon replaces or adds; and the mixture that makes the reorganization subunit has the polymer synthetics of the subunit of the ability of inducing the reaction of anti-Toxins, pertussis immunoprotection in external combination with formation.

5, according to the method for claim 4, wherein the polymer component has the ability of the immunoprotection reaction of inducing anti-Whooping cough disease.

6, according to the method for claim 4, wherein indivedual S2, S3, S4 and S5 subunit are the molar ratio blended with any stoichiometric quantity.

7, according to the method for claim 4, wherein indivedual S2, S3, S4 and S5 subunit are to be about 1: 1: 2: stoichiometrical mixed in molar ratio of 1.

8, according to the method for claim 4 wherein mixture be dissolving reorganization subunit S2, S3, S4 and S5 under reductive condition, and then make it to stand oxidizing condition and form.

9, method according to Claim 8, wherein reductive agent is dithiothreitol (DTT) (DTT).

10, method according to Claim 8, wherein oxygenant is air and Cu ₂SO ₄

11, method according to Claim 8, wherein mixture forms in the presence of solubilizing agent (Chaotrope), stain remover or denaturing agent.

12, according to the method for claim 11, wherein solubilizing agent/denaturing agent is hydrochloric acid Guanidinium.

13, according to the method for claim 11, wherein solubilizing agent/denaturing agent is a urea.

14, according to the method for claim 11, wherein after oxidation, reduce the concentration of solubilizing agent, stain remover or denaturing agent, or change over solubilizing agent, stain remover or the denaturing agent of another kind of lower concentration, be beneficial to the subunit folding and make them be combined into polymer protein.

15, according to the method for claim 14, wherein exchanging solubilizing agent/denaturing agent is hydrochloric acid Guanidinium.

16, according to the method for claim 14, wherein exchanging solubilizing agent/denaturing agent is urea.

17, according to the method for claim 4, wherein heterologous host is the prokaryotic organism bodies.

18, according to the method for claim 17, wherein prokaryotic hosts is intestinal bacteria.

19, according to the method for claim 18, wherein recombinate S2, S3, S4 and S5 subunit are that the form with inclusion body reclaims from intestinal bacteria.

20, according to the method for claim 19, the polymer synthetics purification by chromatography of the subunit of wherein recombinating.

21, according to the method for claim 20, the polymer synthetics affinity chromatography purifying of the subunit of wherein recombinating.

22, according to the method for claim 21, the polymer synthetics of the subunit of wherein recombinating is with fetoprotein affinity chromatography purifying.

23, according to the method for claim 22, the polymer synthetics of the subunit of wherein recombinating is with haptoglobin affinity chromatography purifying.

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