CN102462839B - polysaccharide conjugate vaccine and preparation method thereof - Google Patents
polysaccharide conjugate vaccine and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of polysaccharide conjugate vaccine, comprise the chemical coupling thing of bacterial polysaccharides and carrier protein, described carrier protein is bacterial slime Invasive associated protein, as Heat-labile enterotoxin B, campylobacter jejuni flagellum secretory protein A1, campylobacter jejuni flagellum secretory protein A2, pneumococcal surface protein, Pneumococcal Surface adhesin.The invention still further relates to the preparation method of this polysaccharide conjugate vaccine.Polysaccharide conjugate vaccine of the present invention can under the prerequisite of not adding other any extrinsic protein, and the mucosa that realize polysaccharide conjugate vaccine is delivered, and this immunization route will make more efficient, the convenient and safety of the use of vaccine.
Description
Technical field
The present invention relates to vaccines arts, more specifically, the present invention relates to polysaccharide conjugate vaccine and preparation method thereof.
Background technology
The capsular polysaccharide of antibacterial is a kind of important protective antigen.Chemically the bacterial eapsular polysaccharide component vaccine of purification keeps off infection, it is one of Important Action in Vaccine Development history, wherein in worldwide, the polysaccharide vaccine of extensive use has 4 valency polysaccharide vaccine such as A, C, Y and W135 of epidemic cerebrospinal meningitis coccus, and Diplococcus pneumoniae 23 valency polysaccharide vaccine.But polysaccharide vaccine exists significant limitation, the immunogenicity in 2 years old Infants Below is poor, can not induce immunoprotection effectively.With the vaccine that the polysaccharide (PS) of antibacterial is prepared by chemical method with protein carrier, be referred to as combined vaccine (conjugatevaccine).Early than nineteen twenty-nine, just successfully chemically pneumococcal 3rd type capsular polysaccharide is covalently bond to protein carrier first gets on to be prepared into polysaccharide-protein combined vaccine to Goebel and Avery.This have thymus dependent (T-dependent, TD) protein carrier can by thymus-independent (T-independent, TI) polysaccharide antigen is transformed into the antigen of thymus dependent, thus startup T helper lymphocyte produces a series of immune-enhancing effect.
Drop into since clinical practice through U.S. food and medication management administration (FDA) approval in 1987 from first bloodthirsty hemophilus influenza combined vaccine (Hib) of polysaccharide conjugate vaccine-b type, the research and development of various GL-PP combined vaccine is rapid, has become one of the focus in current vaccine research field.Current combined vaccine mainly contains hemophilus influenza combined vaccine, pneumococcal conjugated vaccine, meningococcus combined vaccine, dysentery bacterium combined vaccine, typhoid conjugate vaccine, beta hemolytic streptococcus combined vaccine etc.
Most polysaccharide conjugate vaccine is mainly using tetanus toxoid (TT) and the endotoxic detoxified mutant of diphtheria (CRM197) as carrier, kind is more single, polysaccharide antibody can be caused to reply low phenomenon, this be due to carrier protein identical in combined vaccine can only with same helper T lymphocyte (Th) competitive binding, thus the B cell quantity making often kind of polysaccharide antigen finally activate reduces, the antibody horizontal of polysaccharide specificity declines.The variation of carrier can make the problems referred to above improve, and its reason may be more than the Th number of cell clones of single carrier protein, will can produce higher levels of specific antibody with the Th number of cell clones activated during different carriers protein binding same polysaccharide.Meanwhile, combined vaccine can produce the immunne response for polysaccharide and protein carrier simultaneously, for the exploitation of multiple vaccines provides thinking.Combined vaccine also has carrier protein effect, and when inoculating combined vaccine, carrier protein can produce the effect of similar adjuvant.Therefore, imperative to the research and development of carrier protein.
Many researcheres are devoted to exploitation mucosa delivery type vaccine, this delivery approach is identical with the natural infection approach of most of pathogen, and mucosa is delivered, and approach is simple, safety, avoids the strict standard needed for vaccinate, is conducive to the immunity inoculation of carrying out large-scale crowd.Much research shows, for some pathogen infected by mucosal route in natural infection, carry out vaccine immunity by mucosal route to body, the immunoprotection that body produces is the most effective.It can produce the local immunity protective reaction of body Mucosal system, and the immunoreation that can produce again general carrys out prophylactic generation.Therefore, develop mucosa delivery type vaccine and there is certain science and necessity.
There are some researches show, be that the C meningococcal polysaccharide protein conjugate vaccines of carrier protein passes through Nasal immunization mice with TT, induce good mucosal immune response.After the oligosaccharide kind material that indecomposable form hemophilus influenza (NTHi) is originated is combined with TT by some researcheres, Nasal immunization mice, result creates good humoral immunization and cellullar immunologic response.Therefore, TT can as the protein carrier of mucosa delivery type GL-PP combined vaccine.
Some pathogen protein ingredients that Heat-labile enterotoxin B (LTB) is representative are the more class mucosal adjuvants of Recent study.Research shows, influenza virus subunit antigen or diphtheria toxoid (DT) form vaccine with LTB, after common intranasal vaccinated mice, in serum, create the specific IgG antibodies of high titre, in oral cavity, vaginal secretions and lung surface create the mucosal IgA antibodies of medium level.
The C meningococcal polysaccharide conjugate of current research mainly with TT or CRM197 for carrier, E.coli LT LT-K63 just strengthens the immunogenicity of conjugate as adjuvant.The bacterial polysaccharides conjugate that to have not yet to see with this albuminoid be carrier protein.Therefore, the polysaccharide carrier albumen simultaneously with protective antigen and adjuvant function to be studied at present and then to improve and the function that extends existing polysaccharide conjugate vaccine becomes focus just day by day.The GL-PP combined vaccine being carrier with this albuminoid has important practical application meaning and wide market prospect.
Summary of the invention
The object of this invention is to provide a kind of polysaccharide conjugate vaccine.
The present invention also aims to the preparation method that this polysaccharide conjugate vaccine is provided.
In order to realize object of the present invention, the invention provides a kind of polysaccharide conjugate vaccine, comprising the chemical coupling thing of bacterial polysaccharides and carrier protein, wherein said carrier protein is bacterial slime Invasive associated protein.
Preferably, described carrier protein is selected from Heat-labile enterotoxin B (LTB), campylobacter jejuni flagellum secretory protein A1 (FspA1), campylobacter jejuni flagellum secretory protein A2 (FspA2), pneumococcal surface protein (PspA) and Pneumococcal Surface adhesin (PsaA).
Preferably, described bacterial polysaccharides is selected from meningococcal polysacharide, bloodthirsty influenzae saccharide and pneumococal polysaccharide.
Preferably, described bacterial polysaccharides is meningococcal polysacharide, and described carrier protein is Heat-labile enterotoxin B.
Vaccine of the present invention can be the form of the liquid preparation that the inventive method directly prepares, and also can be by this liquid by the concentrated concentrated solution form obtained, or the freeze dried powder form by obtaining after dry (such as) lyophilization.
Preferably, described polysaccharide conjugate vaccine is liquid preparation, the free bacteria polysaccharide be not combined with described carrier protein is also included in this liquid preparation, and in described liquid preparation, the content of total bacterial polysaccharides is 30-800 μ g/ml, the content of carrier protein is 30-900 μ g/ml, and total weight ratio between bacterial polysaccharides and carrier protein is 0.3-3.
Preferably, in vaccine of the present invention, the content of the free bacteria polysaccharide be not combined with described carrier protein is lower than 30%.
Preferably, the liquid forms of described polysaccharide conjugate vaccine is that the mode of being delivered by injection or mucosa is used.
The present invention also provides a kind of preparation method of polysaccharide conjugate vaccine, and the method comprises:
1) bacterial polysaccharides is provided;
2) carrier protein is provided;
3) described bacterial polysaccharides and described carrier protein chemical coupling is made;
Wherein, described carrier protein is bacterial slime Invasive associated protein.
Preferably, described carrier protein is selected from Heat-labile enterotoxin B (LTB), campylobacter jejuni flagellum secretory protein A1 (FspA1), campylobacter jejuni flagellum secretory protein A2 (FspA2), pneumococcal surface protein (PspA) and Pneumococcal Surface adhesin (PsaA).
Preferably, described bacterial polysaccharides is selected from meningococcal polysacharide, bloodthirsty influenzae saccharide and pneumococal polysaccharide.
In the present invention, gene engineering method in-vitro recombination expression carrier protein can be utilized, comprise (such as): Heat-labile enterotoxin B (LTB), campylobacter jejuni flagellum secretory protein A1 (FspA1), campylobacter jejuni flagellum secretory protein A2 (FspA2), pneumococcal surface protein (PspA) and Pneumococcal Surface adhesin (PsaA), and carrier protein and polysaccharide antigen are carried out chemical coupling be prepared into GL-PP conjugate.The present inventor finds, this conjugate can as the vaccine of the pathogen corresponding to prevention polysaccharide and albumen.Because these carrier proteins are escherichia coli, campylobacter jejuni and pneumococcal protective antigen composition respectively; it is a kind of protective antigen and stronger mucosal adjuvants; therefore; if using the carrier protein of these albumen as polysaccharide conjugate vaccine, traditional polysaccharide conjugate vaccine can be made to be modified as mucosa delivery type vaccine.And diversified carriers can solve the toxicity cumulative appearance of the low phenomenon of the single antibody response caused of carrier in multivalence polysaccharide conjugate vaccine and carrier protein.Moreover, the polysaccharide conjugate vaccine being carrier with these albumen provides for enterotoxigenic E．Coli, campylobacter jejuni and the reaction of pneumococcal Cross immunogenicity.
Accompanying drawing explanation
Fig. 1 is that recombiant plasmid pET42b+LTB builds schematic diagram (LTB:LTB gene; NdeI:NdeI restriction enzyme site; AvrII:AvrII restriction enzyme site);
Fig. 2 is that recombiant plasmid pET30a+fspA1 builds schematic diagram (fspA1:fspA1 gene; NdeI:NdeI restriction enzyme site; SacI:SacI restriction enzyme site);
Fig. 3 is that recombiant plasmid pET30a+pspA builds schematic diagram (pspA:pspA gene; NdeI:NdeI restriction enzyme site; SacI:SacI restriction enzyme site).
Detailed description of the invention
Below in conjunction with accompanying drawing, by the description of detailed description of the invention, the invention will be further described, but this is not limitation of the present invention, those skilled in the art are according to basic thought of the present invention, various amendment or improvement can be made, but only otherwise depart from basic thought of the present invention, all within the scope of the present invention.
Culture medium used in the present invention is prepared by following method:
LB fluid medium: tryptone (bacto-tryptone, OXOID company) 10g, yeast extract (bacto-yeastextract, OXOID company) 5g, NaCl10g, adding distil water dissolves, be settled to 1000ml, latter 4 DEG C of autoclaving (0.12Mpa, 115 DEG C, 20 minutes) saves backup.
LB fluid medium (Kana+): tryptone (bacto-tryptone, OXOID company) 10g, yeast extract (bacto-yeastextract, OXOID company) 5g, NaCl10g, adding distil water dissolves, and is settled to 1000ml, autoclaving (0.12Mpa, 115 DEG C, 20 minutes), when temperature drops to 50 DEG C by the time, adding kanamycin (Kana) to final concentration is 30ug/ml, and 4 DEG C save backup.
LB solid medium (Kana+): add 1.5g agar in the above-mentioned LB fluid medium of 100ml, autoclaving (0.12Mpa, 115 DEG C, 20 minutes), by the time when temperature drops to 50 DEG C, adding kanamycin (Kana) to final concentration is 30ug/ml, bed board, and 4 DEG C save backup.
Embodiment 1
Be combined into example below with Heat-labile enterotoxin B (LTB) and C group's epidemic cerebrospinal meningitis Streptococcus polysaccharides (GCMP), come that the present invention is described in detail.
(1), the cloning and expressing 1 of recombination bacillus coli heat labile enterotoxin B subunit (rLTB)) synthesis of LTB gene and the structure of expression vector
LTB gene order is selected from GENBANK, and serial number is GI:157021177.According to e. coli codon frequency of utilization table (list of references: Maloy, S., V.Stewart, andR.Taylor.1996.Geneticanalysisofpathogenicbacteria.Col dSpringHarborLaboratoryPress, NY.) this sequence is optimized, add 6 histidine-tagged and termination codoies at sequence C end, then add NdeI, AvrII restriction enzyme site respectively at sequence two ends.
The above-mentioned LTB gene of synthetic, then uses NdeI, AvrII double digestion LTB and pET42b (purchased from Novagen), then the object fragment after double digestion LTB is connected with pET42b carrier, is built into recombiant plasmid pET42b+LTB, as shown in Figure 1.Full genome synthesis and above-mentioned construction work are completed by Shanghai Jierui Biology Engineering Co., Ltd.
2) the enzyme action qualification of recombinant vector
By step 1) the recombiant plasmid pET42b+LTB that obtains is transformed in bacillus coli DH 5 alpha, and transformant is inoculated on LB solid medium (Kana+) flat board, the bacterium colony that therefrom picking growth conditions is good, be connected in LB fluid medium (Kana+), in 37 DEG C of shaking tables, 230rpm incubated overnight.Extract plasmid, carry out NdeI, AvrII double digestion, through agarose gel electrophoresis analytical proof, have LTB genes of interest fragment and pET42b carrier segments on a corresponding position, stripe size is consistent with expection.
3) expression of destination protein LTB
By step 1) the recombiant plasmid pET42b+LTB that obtains is converted in e. coli bl21, when to be cultured to OD value in 237rpm, 37 DEG C of incubators be 1.0, add the isopropylthiogalactoside (IPTG) that final concentration is 1mM, abduction delivering 2-4 hour (list of references: " molecular cloning texts guide " third edition (upper and lower volume) (U.S.) J. Pehanorm Brooker, Huang Peitang, Science Press, 2002).
(list of references: " molecular cloning texts guide " is detected through SDS-PAGE, ibid) and Westernblot qualification (list of references: " molecular cloning texts guide ", ibid) prove, the expression product of above-mentioned e. coli bl21 is LTB, and can with the monoclonal antibody of LTB (purchased from HyTest company) specific binding, conform to expected results.
(2), the purification of rLTB
By centrifugal for the e. coli bl21 after above-mentioned abduction delivering (5000rpm, 10 minutes), abandon supernatant, the phosphate buffer (sodium dihydrogen phosphate-sodium hydrogen phosphate) of thalline with 20mM, pH7.0 is cleaned once, resuspended with the phosphate buffer of above-mentioned 20mM, pH7.0 again, broken with Ultrasonic Cell Disruptor.Broken completely rear centrifugal (11000rpm, 40 minutes), collect supernatant, with CM-FF cation exchange column (purchased from GEHEALTHY) purification, be collected in eluting peak when eluent is 0.17mol/LNaCl, obtain the object of the invention albumen rLTB after purification.
(3), the chemical coupling of rLTB and GCMP
After being dialysed 48 hours at 4 DEG C by above-mentioned purification gained albumen rLTB, filtration sterilization, saves backup in 4 DEG C.Get 2.5mlGCMP (present of Tiantan Bio-pharmaceuticals goods limited company) (about 10mg), the CNBr of quality such as to add, stir and react, and maintain pH10.8 with 0.1MNaOH, under high ph conditions, the hydroxyl reaction on CNBr and polysaccharide sugar chain generates cyanate.After above-mentioned reaction carries out 10 minutes, add adipic dihydrazide (ADH) 70mg, maintain pH8.5 with 0.1MNaOH, spend the night in 4 DEG C of reactions, make ADH and polyisocyanate reactant form isourea key, generate GCMP-ADH hydrazide derivative.Then reactant mixture is loaded bag filter, dialyse 48 hours in 0.2MNaCl, remove unreacted CNBr, ADH or reaction intermediates.Then the pH value to 5.7 of dialysate is regulated with hydrochloric acid, add above-mentioned obtained carrier protein rLTB, make dialysate (polysaccharide) equal with the quality of rLTB, add final concentration 0.1MEDAC (carbodiimide) again, maintain pH5.7 with hydrochloric acid, stir dialysis 2 days in 2-8 DEG C, make the carboxyl of carrier protein and the amino generation condensation reaction of GCMP-ADH hydrazide derivative, generate GCMP-ADH-carrier protein, then collect dialysate.
After above-mentioned cyanate and ADH react the 48h that dialyses, with the content of ADH in TNBS colorimetric determination derivant, can reflect the average degree of modification of polysaccharide, testing result is 0.8-1.5% (weight ratio).
(4), the purification of the conjugate of rLTB and GCMP
Product step (three) obtained is ultrafiltration and concentration in 10kD super filter tube (Millipore), use 0.45 μm of membrane filtration again, purify with Sephorose4FF gel column, use 0.2MNaCl is eluant, collect the eluting peak at void volume place, obtain GCMP-rLTB conjugate solution of the present invention.Qualitative detection and detection by quantitative are carried out to GCMP-rLTB conjugate, wherein according to having at molecular sieve void volume place 280nm wavelength place, carry out qualitative detection without absworption peak to GCMP-rLTB conjugate, then show that carrier protein and polysaccharide generate conjugate if any absworption peak, otherwise then illustrate do not have GL-PP conjugate to generate.Method described in the Pharmacopoeia of the People's Republic of China the 3rd (version in 2010, Chinese Pharmacopoeia Commission compiles, China Medical Science Press) is adopted to the detection of polysaccharide in conjugate and protein content.The result of described qualitative detection and detection by quantitative is: at molecular sieve void volume place 280nm wavelength, there is absworption peak at place, protein content: 246.5 μ g/ml, total polysaccharides content: 640.1 μ g/ml, dissociation amylase content: 15.1 μ g/ml, polysaccharide/albumen (weight ratio): 2.5.
(5), the immune effect of GCMP-rLTB conjugate
1. test drug: the GCMP-rLTB conjugate prepared by said method, and GCMP-TT (present of Bacterial Laboratory of Beijing Biological Product Inst.), GAMP-TT+GCMP-TT (present of Bacterial Laboratory of Beijing Biological Product Inst.), GCMP (present of Tiantan Bio-pharmaceuticals goods limited company), and be mixed with the liquid preparation with the required total polysaccharides content of test.
2. experimental animal: BALB/c mouse, 24, female, in 6 week age, body weight 18-20g, purchased from Tiantan Bio-pharmaceuticals company, raises at Tiantan Bio-pharmaceuticals company Animal House.
3. animal grouping: by BALB/c mouse at random every 6 be divided into one group.
4. immunization ways:
Collunarium group 34 μ l/ only, is 10 μ g or 20ug containing total polysaccharides, slowly instills from mice nostril, notes not flowing into oral cavity and lung;
Lumbar injection group 100 μ l/, containing total polysaccharides 10 μ g or 20ug;
Immunity 4 times altogether, immunity in the 0th, 14,21 day, namely rinses mouse vagina/oral cavity/lung/small intestinal with containing 2% saponin PBS (pH7.4) and after collecting flushing liquor, gets blood about 500 μ l detect at eyeball of mouse place for after third time immunity 1 week on the 21st day.Namely, after within after final immunization 1 week, within the 28th day, collecting flushing liquor in the same way, pluck eyeball and get blood and draw neck to put to death mice.
5. detection method:
The ELISA that mouse vagina/oral cavity/lung/small intestinal rinses anti-polysaccharide IgA in thing measures: after being spent the night by 96 hole ELISA Plate 4 DEG C with every hole 100 μ l diluent (containing total polysaccharides 10 μ g) bag, 37 DEG C of closed 3h, after 1: 32 initial dilution mouse vagina/oral cavity/lung/small intestinal flushing liquor, carry out doubling dilution again, add in hand-hole with every hole 100 μ l, be placed in 37 DEG C of incubator 1h; Add with the sheep anti mouse IgA100 μ l/ hole of 1: 2000 dilution, develop the color after being placed in 37 DEG C of incubator 1h and use microplate reader reading.
In mouse blood, the ELISA of anti-polysaccharide IgG measures: after spending the night with every hole 100 μ l diluent (containing total polysaccharides 10 μ g) coated elisa plate 4 DEG C, 37 DEG C of closed 3h, serum after centrifugal with 1: 100 initial dilution, carry out doubling dilution again, add in hand-hole with every hole 100 μ l, be placed in 37 DEG C of 1h; Add with the sheep anti-mouse igg 100 μ l/ hole of 1: 5000 dilution, develop the color after being placed in 37 DEG C of incubator 1h and use microplate reader reading.
The ELISA that mouse small intestine/lung rinses anti-LTBIgA in thing measures: after being spent the night with the concentration coated elisa plate of 10ug/ml (every hole 100 μ l) 4 DEG C by LTB albumen (available from Sigma), 37 DEG C of closed 3h, thing is rinsed with 1: 10 doubling dilution small intestinal/lung, add in hand-hole with every hole 100 μ l, hatch 1h for 37 DEG C; Add with the sheep anti mouse IgA of 1: 2000 dilution, develop the color after being placed in 37 DEG C of incubator 1h and use microplate reader reading.
In mouse blood, the ELISA of anti-LTBIgG measures: after being spent the night with the concentration coated elisa plate of 10ug/ml (every hole 100 μ l) 4 DEG C by LTB albumen (available from Sigma), 37 DEG C of closed 3h, with 1: 40 doubling dilution serum, add in hand-hole with every hole 100 μ l, be placed in 37 DEG C of 1h; Add with the sheep anti-mouse igg of 1: 5000 dilution, develop the color after being placed in 37 DEG C of incubator 1h and use microplate reader reading.
6. result of the test:
a.GCMP specific serum and mucosal immune response level
When testing 1. Nasal immunization GCMP-rLTB specific serum and mucosal immune response level and with the comparing of GCMP-TT immunne response level
Result of the test is in table 1.As can be seen from Table 1, the GCMP-rLTB Nasal immunization mice that total polysaccharides is 10 μ g or 20ug is contained by using, 1 week after the 4th immunity is 28 days, and it 1 week is all polysaccharide antibody titre (P < 0.01) in the 21st Tianchong washing that mouse vagina rinses polysaccharide antibody titre in thing after third time immunity; In blood, polysaccharide antibody titre is also all apparently higher than the rear 1 week antibodies in blood titre (P < 0.01) of third time immunity; And 1 week after the 4th immunity is 28 days, murine oral rinses in thing and lung flushing thing and also all shows a certain amount of polysaccharide antibody titre.
In addition, GCMP-rLTB collunarium group is compared with GCMP-TT collunarium group, and the antibody titer that mouse vagina/oral cavity/lung rinses the anti-polysaccharide IgA in thing and the anti-polysaccharide IgG in blood has significant difference (P < 0.05).This result illustrates that LTB and TT two kinds of carrier proteins are with on the coupling effect of GCMP, and the former is better than the latter.
The Efficacy evaluation data (GMT is geometric mean) of table 1GCMP-rLTB conjugate, GCMP-TT
The comparison of the specific serum immunne response level that test 2.GCMP-rLTB, GCMP-TT and GCMP are produced by different immunization route
(1) comparison of specific serum immunne response level that produced by lumbar injection approach of .GCMP-rLTB and GCMP-TT
By using GCMP-TT and containing the GCMP-rLTB lumbar injection immune mouse that total polysaccharides is 10 μ g, 1 week after the 4th immunity is 28 days, and detect anti-polysaccharide IgG in mouse blood, result of the test is in table 2.As can be seen from Table 2, GCMP-rLTB lumbar injection group is compared with GCMP-TT lumbar injection group, specific serum GCMPIgG antibody horizontal has significant difference (P < 0.05), and the antibody horizontal that the former produces is about 5.5 times of the latter.This result illustrates that rLTB and TT two kinds of protein carriers are with on the coupling effect of GCMP, and the former is better than the latter.Due to method of drawing material, GCMP-TT lumbar injection group could not detect IgA antibody at lung and intestinal.
The Efficacy evaluation data (GMT is geometric mean) of table 2GCMP-rLTB conjugate, GCMP-TT
(2) comparison of the immunne response level of .GCMP-rLTB lumbar injection group and the generation of GCMP-rLTB collunarium group
By use containing total polysaccharides be the GCMP-rLTB of 10 μ g respectively by lumbar injection and Nasal immunization mice, namely after the 4th immunity 1 week 28 days, detect anti-polysaccharide IgG in mouse blood, small intestinal rinses thing IgA and lung flushing thing IgA, result of the test was in table 2.As can be seen from Table 2, GCMP-rLTB lumbar injection group is compared with both GCMP-rLTB collunarium groups, serological specificity GCMPIgG antibody horizontal there was no significant difference, but the antibody horizontal that the latter produces is about the former 2.5 times, lung rinses the horizontal there was no significant difference of IgA antibody in thing and small intestinal flushing thing, but the antibody horizontal of collunarium group is still higher than injection group, this result shows that rLTB has played the function of carrier and mucosal adjuvants simultaneously.
(3) the immunne response level that .GCMP and GCMP-rLTB is produced by lumbar injection approach compares
By using GCMP and containing the GCMP-rLTB lumbar injection immune mouse that total polysaccharides is 10 μ g, 1 week after the 4th immunity is 28 days, and detect anti-polysaccharide IgG in mouse blood, small intestinal rinses thing IgA and lung rinses thing IgA, result of the test is in table 2.As can be seen from Table 2, immunity is carried out by using GCMP-rLTB, the serological specificity IgG antibody level produced, lung rinse thing all has significant difference (P < 0.05) with the IgA antibody level that small intestinal rinses in thing compared with simple GCMP, and this result shows that LTB has the great potential as polysaccharide conjugate vaccine novel carriers.
(4) the immunne response level that .GAMP-TT+GCMP-TT and GAMP-TT+GCMP-rLTB is produced by lumbar injection approach compares
By using GAMP-TT+GCMP-TT and GAMP-TT+GCMP-rLTB (be 10 μ gs containing total polysaccharides) lumbar injection immune mouse, after the 4th immunity 1 week namely 28 days, GAMP-IgG and GCMP-IgG in detection mouse blood, result of the test was in table 3.As can be seen from Table 3, the serological specificity GCMPIgG antibody horizontal of two groups has significant difference (P < 0.05), the antibody horizontal of the latter is about the former 30 times, serological specificity GAMPIgG antibody horizontal has significant difference (P < 0.05), the antibody horizontal of the latter is about the former 3.6 times, and this result shows to select different albumen to be more better than single protein carrier as the immune effect of carrier in multivalence polysaccharide conjugate vaccine simultaneously.
Polysaccharide specificity Serological IgG level (GMT is geometric mean) after the immunity of table 3A, C group polysaccharide conjugate lumbar injection
b.LTB specific serum and mucosal immune response level
Result of the test is in table 4.As can be seen from Table 4, containing total polysaccharides by use is that the GCMP-rLTB of 10 μ g is through collunarium or lumbar injection immune mouse, 1 week after the 4th immunity is 28 days, in the serum of mice, produce the IgG antibody of the anti-LTB of relative high levels, in the mucosal tissue (small intestinal rinses thing, lung rinses thing) of small intestinal, produce the IgA antibody of relatively low anti-LTB.And lumbar injection group compares with collunarium group, serum and mucosa specific antibody level all have significant difference (P < 0.05), but the antibody level of serum that injection group produces is higher than collunarium group, and the mucoantibody level that collunarium group produces is higher than injection group, illustrates that rLTB is delivered by mucosa and can produce good mucosal immune response.
Table 4LTB specific serum and mucosal immune response level (GMT is geometric mean)
Embodiment 2
Be combined into example below with campylobacter jejuni flagellum secretory protein A1 (FspA1) and A group's epidemic cerebrospinal meningitis ball polysaccharide (GAMP), come that the present invention is described in detail.
(1), the cloning and expressing of FspA1
1) synthesis of FspA1 gene and the structure of expression vector
FspA1 gene order is selected from GENBANK, and serial number is GI:116292639.According to e. coli codon frequency of utilization table (list of references: Maloy, S., V.Stewart, andR.Taylor.1996.Geneticanalysisofpathogenicbacteria.Col dSpringHarborLaboratoryPress, NY.) this sequence is optimized, add 6 histidine-tagged and termination codoies at sequence C end, then add NdeI, SacI restriction enzyme site respectively at sequence two ends.
The above-mentioned FspA1 gene of synthetic, then NdeI, SacI double digestion FspA1 and pET30a (purchased from Novagen) is used, again the object fragment after double digestion FspA1 is connected with pET30a large fragment, is built into recombiant plasmid pET30a+FspA1, as shown in Figure 2.Full genome synthesis and above-mentioned construction work are completed by Shanghai Jierui Biology Engineering Co., Ltd.
2) the enzyme action qualification of recombinant vector
By step 1) the recombiant plasmid pET30a+FspA1 that obtains is transformed in e. coli bl21, and transformant is inoculated on LB solid medium (Kana+) flat board, the bacterium colony that therefrom picking growth conditions is good, be connected in LB fluid medium (Kana+), in 37 DEG C of shaking tables, 230rpm incubated overnight.Extract plasmid, carry out NdeI, SacI double digestion, through agarose gel electrophoresis analytical proof, have FspA1 genetic fragment and pET30a+ plasmid on a corresponding position, consistent with expection.
3) expression of destination protein PspA
By step 1) the recombiant plasmid pET30a+FspA1 that obtains is converted in e. coli bl21, when to be cultured to OD value in 237rpm, 37 DEG C of incubators be 1.0, add the isopropylthiogalactoside (IPTG) that final concentration is 1mM, abduction delivering 2-4 hour (list of references: " molecular cloning texts guide ", ibid).
(list of references: " molecular cloning texts guide " is detected through SDS-PAGE, ibid) and Westernblot qualification (list of references: " molecular cloning texts guide ", ibid) prove, the expression product of above-mentioned e. coli bl21 is FspA1, and with histidine-tagged, can with polyhistidine antibody specific binding, conform to expected results.
(2), the purification of FspA1
By centrifugal for the e. coli bl21 after above-mentioned abduction delivering (5000rpm, 10 minutes), abandon supernatant, the phosphate buffer (sodium dihydrogen phosphate-sodium hydrogen phosphate) of thalline with 20mM, pH7.5 is cleaned once, resuspended with the phosphate buffer of above-mentioned 20mM, pH7.5 again, broken with Ultrasonic Cell Disruptor.Broken completely rear recentrifuge (11000rpm, 40 minutes), collect supernatant, with HisTrapFF post (purchased from GEHEALTHY) purification, be collected in eluting peak when eluent is 0.250M imidazoles, 0.5MNaCl, 20mMPB, obtain the object of the invention albumen FspA1 after purification.
(3), the coupling of FspA1 and GAMP
After being dialysed 48 hours at 4 DEG C by above-mentioned purification gained albumen FspA1, filtration sterilization, saves backup in 4 DEG C.Get 2.5mlGCMP (present of Tiantan Bio-pharmaceuticals goods limited company) (about 10mg), the CNBr of quality such as to add, stir and react, and maintain pH10.5 with 0.1MNaOH, under high ph conditions, the hydroxyl reaction on CNBr and polysaccharide sugar chain generates cyanate.After above-mentioned reaction carries out 10 minutes, add adipic dihydrazide (ADH) 70mg, maintain pH8.5 with 0.1MNaOH, spend the night in 4 DEG C of reactions, make ADH and polyisocyanate reactant form isourea key, generate GCMP-ADH hydrazide derivative.Then reactant mixture is loaded bag filter, dialyse 48 hours in 0.2MNaCl, remove unreacted CNBr, ADH or reaction intermediates.Then the pH value to 6.7 of dialysate is regulated with hydrochloric acid, add above-mentioned obtained carrier protein FspA1, make dialysate (polysaccharide) equal with the quality of FspA1, add final concentration 0.1MEDAC (carbodiimide) again, maintain pH5.5 with hydrochloric acid, react 3 hours, make the carboxyl of carrier protein and the amino generation condensation reaction of GCMP-ADH hydrazide derivative, generate GCMP-ADH-carrier protein, then collect dialysate.
After above-mentioned cyanate and ADH react the 48h that dialyses, with the content of ADH in TNBS colorimetric determination derivant, can reflect the average degree of modification of polysaccharide, testing result is 0.8-1.5% (weight ratio).
(4), the purification of the conjugate of FspA1 and GAMP
The product that step (three) is obtained ultrafiltration 5-7 time in 0.2MNaCl super filter tube, use 0.45 μm of membrane filtration again, purify with Sephorose4FF gel column, use 0.2MNaCl is eluant, collect the eluting peak at void volume place, obtain GAMP-FspA1 conjugate solution of the present invention.Carry out qualitative detection and detection by quantitative to GCMP-FspA1 conjugate, method is with embodiment 1, and result is: at molecular sieve void volume place 280nm wavelength, there is absworption peak at place, protein content 868.21 μ g/ml; Total polysaccharides content 301.8568 μ g/ml; Dissociation amylase content 16.05%; Polysaccharide/albumen (weight ratio) 0.348.
(5), the Efficacy evaluation of conjugate
1. test drug: the GAMP-FspA1 conjugate prepared by said method, and be mixed with the liquid preparation with the required total polysaccharides content of test.
2. experimental animal: with embodiment 1.
3. animal grouping: 24 BALB/c mouse are divided at random 4 groups (often organizing 6): normal saline collunarium group, normal saline lumbar injection group, FspA1-GAMP conjugate collunarium group, FspA1-GAMP conjugate lumbar injection group.
4. immunization ways:
Collunarium group 34 μ l/ only, is 10 μ g containing total polysaccharides, slowly instills from mice nostril, notes not flowing into oral cavity and lung;
Lumbar injection group 100 μ l/, containing total polysaccharides 10 μ g;
Normal saline collunarium group and lumbar injection group respectively instill 34 μ l respectively and are injected into 100 μ l normal saline.
Altogether immunity 4 times, immunity in the 0th, 14,21 day, namely rinses mouse vagina with the PBS (pH7.4) containing 2% saponin on the 21st day in after third time immunity 1 week and after collecting flushing liquor, gets blood about 500 μ l detect at eyeball of mouse place.Namely, after within after final immunization 1 week, within the 28th day, collecting flushing liquor in the same way, pluck eyeball and get blood and draw neck to put to death mice.
5. detection method:
The ELISA that mouse vagina rinses anti-polysaccharide IgA in thing measures: with embodiment 1.
In mouse blood, the ELISA of anti-polysaccharide IgG measures: with embodiment 1.
6. result of the test:
Result of the test is in table 5.As can be seen from Table 5,1 week after the 4th immunity is 28 days, and it 1 week is polysaccharide antibody titre (P < 0.01) in the 21st Tianchong washing that mouse vagina rinses polysaccharide antibody titre in thing after third time immunity; In blood, polysaccharide antibody titre is also apparently higher than the rear 1 week antibodies in blood titre (P < 0.01) of third time immunity; The antibody titer value of normal saline collunarium group, normal saline lumbar injection group is zero.This result explanation FspA1 can as the good carrier of the polysaccharide conjugate vaccine of mucosal immunity.In addition, the campylobacter jejuni of bibliographical information campylobacter jejuni flagellum secretory protein A to different serotype is had to have certain immune protective efficiency, can as a kind of candidate albumen of jejunum campylobacter bacteria vaccine.Take FspA1 as the epidemic encephalitis polysaccharide conjugate vaccine of the mucosal immunity of carrier, both can produce the antibody of anti-current brain polysaccharide, also can produce the protection antibody of certain anti-campylobacter jejuni.
Embodiment 3
Be combined into example below with Pneumococcal Surface memebrane protein A (PspA) and A group's epidemic cerebrospinal meningitis ball polysaccharide (GAMP), come that the present invention is described in detail.
(1), the cloning and expressing of restructuring PspA
1) synthesis of PspA gene and the structure of expression vector
PspA gene order is selected from GENBANK, and serial number is GI:193804931.According to e. coli codon frequency of utilization table (list of references: Maloy, S., V.Stewart, andR.Taylor.1996.Geneticanalysisofpathogenicbacteria.Col dSpringHarborLaboratoryPress, NY.) this sequence is optimized, add 6 histidine-tagged and termination codoies at sequence C end, then add NdeI, SacI restriction enzyme site respectively at sequence two ends.
The above-mentioned PspA gene of synthetic, then NdeI, SacI double digestion PspA and pET30a (purchased from Novagen) is used, again the object fragment after double digestion PspA is connected with pET30a large fragment, is built into recombiant plasmid pET30a+PspA, as shown in Figure 3.Full genome synthesis and above-mentioned construction work are completed by Shanghai Jierui Biology Engineering Co., Ltd.
2) the enzyme action qualification of recombinant vector
By step 1) the recombiant plasmid pET30a+PspA that obtains is transformed in e. coli bl21, and transformant is inoculated on LB solid medium (Kana+) flat board, the bacterium colony that therefrom picking growth conditions is good, be connected in LB fluid medium (Kana+), in 37 DEG C of shaking tables, 230rpm incubated overnight.Extract plasmid, carry out NdeI, SacI double digestion, through agarose gel electrophoresis analytical proof, have PspA genetic fragment and pET30a+ plasmid on a corresponding position, consistent with expection.
3) expression of destination protein PspA
By step 1) the recombiant plasmid pET30a+PspA that obtains is converted in e. coli bl21, when to be cultured to OD value in 237rpm, 37 DEG C of incubators be 1.0, add the isopropylthiogalactoside (IPTG) that final concentration is 1mM, abduction delivering 2-4 hour (list of references: " molecular cloning texts guide ", ibid).
(list of references: " molecular cloning texts guide " is detected through SDS-PAGE, ibid) and Westernblot qualification (list of references: " molecular cloning texts guide ", ibid) prove, the expression product of above-mentioned e. coli bl21 is PspA, and with histidine-tagged, can with polyhistidine antibody specific binding, conform to expected results.
(2), the purification of PspA
By centrifugal for the e. coli bl21 after above-mentioned abduction delivering (5000rpm, 10 minutes), abandon supernatant, the phosphate buffer (sodium dihydrogen phosphate-sodium hydrogen phosphate) of thalline with 20mM, pH7.5 is cleaned once, resuspended with the phosphate buffer of above-mentioned 20mM, pH7.5 again, broken with Ultrasonic Cell Disruptor.Broken completely rear recentrifuge (11000rpm, 40 minutes), collect supernatant, with HisTrapFF post (purchased from GEHEALTHY) purification, be collected in eluting peak when eluent is 0.050M imidazoles, 0.5MNaCl, 20mMPB, obtain the object of the invention albumen PspA after purification.
(3), the coupling of PspA and GAMP
After being dialysed 48 hours at 4 DEG C by above-mentioned purification gained albumen PspA, filtration sterilization, saves backup in 4 DEG C.Get 2.5mlGCMP (present of Tiantan Bio-pharmaceuticals goods limited company) (about 10mg), the CNBr of quality such as to add, stir and react, and maintain pH10.5 with 0.1MNaOH, under high ph conditions, the hydroxyl reaction on CNBr and polysaccharide sugar chain generates cyanate.After above-mentioned reaction carries out 10 minutes, add adipic dihydrazide (ADH) 70mg, maintain pH8.5 with 0.1MNaOH, spend the night in 4 DEG C of reactions, make ADH and polyisocyanate reactant form isourea key, generate GCMP-ADH hydrazide derivative.Then reactant mixture is loaded bag filter, dialyse 48 hours in 0.2MNaCl, remove unreacted CNBr, ADH or reaction intermediates.Then the pH value to 6.7 of dialysate is regulated with hydrochloric acid, add above-mentioned obtained carrier protein PspA, make dialysate (polysaccharide) equal with the quality of PspA, add final concentration 0.1MEDAC (carbodiimide) again, maintain pH6.0-6.3 with hydrochloric acid, react 3 hours, make the carboxyl of carrier protein and the amino generation condensation reaction of GCMP-ADH hydrazide derivative, generate GCMP-ADH-carrier protein, then collect dialysate.
After above-mentioned cyanate and ADH react the 48h that dialyses, with the content of ADH in TNBS colorimetric determination derivant, can reflect the average degree of modification of polysaccharide, testing result is 0.8-1.5% (weight ratio).
(4), the purification of the conjugate of PspA and A group epidemic encephalitis polysaccharide (GAMP)
Product step 3 obtained is ultrafiltration and concentration in 10kD super filter tube, use 0.45 μm of membrane filtration again, purify with Sephorose4FF gel column, use 0.2MNaCl is eluant, collect the eluting peak at void volume place, obtain GAMP-PspA conjugate of the present invention.Carry out qualitative detection and detection by quantitative to GCMP-PspA conjugate, method is with embodiment 1, and result is: at molecular sieve void volume place 280nm wavelength, there is absworption peak at place, protein content 433.08 μ g/ml; Total polysaccharides content 372.45 μ g/ml; Dissociation amylase content 11.91%; Polysaccharide/albumen (weight ratio) 0.86.
(5), the Efficacy evaluation of conjugate
1. test drug: the GAMP-PspA conjugate prepared by said method, and be mixed with the liquid preparation with the required total polysaccharides content of test.
2. experimental animal: with embodiment 1.
3. animal grouping: 24 BALB/c mouse are divided at random 4 groups (often organizing 6): normal saline collunarium group, normal saline lumbar injection group, PspA-GAMP conjugate collunarium group, PspA-GAMP conjugate lumbar injection group.
4. immunization ways: with embodiment 2.
5. detection method: with embodiment 2.
6. result of the test:
Result of the test is in table 5.As can be seen from Table 5, after 4th immunity 1 week is 28 days, it immune latter 1 week is the titre (P < 0.01) of polysaccharide antibody in the 21st Tianchong washing apparently higher than third time that mouse vagina rinses polysaccharide antibody titre in thing, and in serum, polysaccharide antibody titre is also apparently higher than the rear 1 week antibodies in blood titre (P < 0.01) of third time immunity; The antibody titer value of normal saline collunarium group, normal saline lumbar injection group is zero.This result illustrates can as the good carrier of polysaccharide conjugate vaccine with streptococcus pneumoniae outer membrane protein A.In addition, the streptococcus pneumoniae of bibliographical information Pneumococcal Surface memebrane protein to different serotypes is had to have certain immune protective efficiency, can as a kind of candidate albumen of Pnu-Imune 23.Mucosa delivery type epidemic encephalitis polysaccharide conjugate vaccine using this kind of albumen as carrier, can produce the antibody of anti-current brain polysaccharide, also can create antagonism pneumococcal certain protection antibody.
Table 5FspA1-GAMP and PspA-GAMP Efficacy evaluation data
In addition; campylobacter jejuni flagellum secretory protein A2 and campylobacter jejuni flagellum secretory protein A1 has the homology of 44% on aminoacid sequence; both protections have certain difference; adhere to invasion and attack relevant with the motion of campylobacter jejuni; also certain cross-protection is had between different serotypes; equally also can, as the carrier of the epidemic encephalitis combined vaccine of mucosal immunity, act on identical with example 2.PsaA pneumococcal surface adhesion A (PsaA) is a kind of adhesion factor; belong to the transport protein of ABC type; cross protection is had between different serotypes; there is report PsaA antibody intranasal immunisations can reduce the field planting of streptococcus pneumoniae at nasopharynx part; certain protection antibody can be produced in mucosal sites; the epidemic encephalitis polysaccharide conjugate vaccine of the mucosa delivery type using streptococcus pneumoniae flagellum adhesion A as carrier both can produce the antibody compared with high resistance epidemic encephalitis polysaccharide, also can produce pneumococcal certain protection antibody.
Claims (6)
1. a polysaccharide conjugate vaccine, is characterized in that, comprises the chemical coupling thing of bacterial polysaccharides and carrier protein, and wherein said carrier protein is bacterial slime Invasive associated protein,
Wherein, described carrier protein is selected from Heat-labile enterotoxin B, campylobacter jejuni flagellum secretory protein A1 or campylobacter jejuni flagellum secretory protein A2;
Wherein, described bacterial polysaccharides is selected from meningococcal polysacharide;
Wherein, described polysaccharide conjugate vaccine is liquid preparation, the free bacteria polysaccharide be not combined with described carrier protein is also included in this liquid preparation, and in described liquid preparation, the content of total bacterial polysaccharides is 30-800 μ g/ml, the content of carrier protein is 30-900 μ g/ml, and total weight ratio between bacterial polysaccharides and carrier protein is 0.3-3.
2. polysaccharide conjugate vaccine according to claim 1, is characterized in that, described bacterial polysaccharides is meningococcal polysacharide, and described carrier protein is Heat-labile enterotoxin B.
3. polysaccharide conjugate vaccine according to claim 1, is characterized in that, in described vaccine, the content of the described free bacteria polysaccharide be not combined with described carrier protein is lower than 30%.
4. polysaccharide conjugate vaccine according to claim 1, is characterized in that, described polysaccharide conjugate vaccine is that the mode of being delivered by injection or mucosa is used.
5. a preparation method for polysaccharide conjugate vaccine according to claim 1, the method comprises:
1) described bacterial polysaccharides is provided;
2) described carrier protein is provided; With
3) described bacterial polysaccharides and described carrier protein chemical coupling is made.
6. method according to claim 5, is characterized in that, described carrier protein is selected from Heat-labile enterotoxin B; Described bacterial polysaccharides is selected from meningococcal polysacharide.
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| CN101072586A (en) * | 2004-09-21 | 2007-11-14 | 圣诺菲·帕斯图尔公司 | Multivalent meningococcal derivatized polysaccharide-protein conjugates and vaccine |
| CN101610785A (en) * | 2006-12-22 | 2009-12-23 | 惠氏公司 | Multivalent pneumococcal polysaccharide-protein conjugate composition |
| CN101780272A (en) * | 2009-12-24 | 2010-07-21 | 湖南农业大学 | Application of autovaccine preparation containing TGF beta 1 |
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| CN101072586A (en) * | 2004-09-21 | 2007-11-14 | 圣诺菲·帕斯图尔公司 | Multivalent meningococcal derivatized polysaccharide-protein conjugates and vaccine |
| CN101610785A (en) * | 2006-12-22 | 2009-12-23 | 惠氏公司 | Multivalent pneumococcal polysaccharide-protein conjugate composition |
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