CN104888209A - B-group epidemic neisseria meningitidis recombinant protein vaccine and preparing method thereof - Google Patents
B-group epidemic neisseria meningitidis recombinant protein vaccine and preparing method thereof Download PDFInfo
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Abstract
The invention provides a B-group epidemic neisseria meningitidis recombinant protein vaccine and a preparing method thereof, and belongs to the field of biological products. The vaccine contains three mutant recombinant proteins of a human H factor binding protein. The invention further provides a preparing method of the B-group epidemic neisseria meningitidis recombinant protein vaccine. The method includes the steps of human H factor binding protein V1, V2 and V3 recombinant expression carrier construction, expression strain screening and identification, fermental cultivation, recombinant protein antigen inducible expression and recombinant protein antigen purification and protein detection; and according to the concentrations of the three antigen components, quantified mixing is carried out, and the B-group epidemic neisseria meningitidis recombinant protein vaccine is prepared in cooperation with an aluminum adjuvant. By means of the B-group epidemic neisseria meningitidis recombinant protein vaccine, intraperitoneal injection is carried out on an immune mice, the mice body can be stimulated to generate a B-group epidemic neisseria resisting peculiar antibody, and the alexin mediated serum sterilization antibody can rise by more than 8 times.
Description
Technical field
The present invention relates to field of biological product, particularly, relate to a kind of B group's epidemic cerebrospinal meningitis coccus recombinant protein vaccine and preparation method thereof.
Background technology
Neisseria meningitidis is the Main Pathogenic Bacteria of bacterial meningitis and pyemia, according to meningococcus phage surface capsular polysaccharide structure and antigenic difference, meningococcus is divided into 13 sero-groups (Serogroup), wherein 5 group (A, B, C, W135 and Y group) be main pathogenic flora.The meningitis sickness rate that the whole world is caused by meningococcus reaches 30-50 ten thousand every year, wherein, about has the infected of 50% to cause primarily of B group meningitis cocci.In recent years, due to antibiotic abuse, occur Resistant strain successively all over the world, this adds larger difficulty to the treatment of disease.
People generally believe and inoculate the ideal measure that vaccine is safely and effectively prevention and corntrol epidemic encephalitis; cross protection is not had between each pathogenic serotypes of meningococcus; inoculation univalent vaccine then can not effectively prevent by the infection of other serological type strain; have multiple unit price, bivalence and tetravalence epidemic encephalitis polysaccharide, the list marketings of GL-PP combined vaccine such as A group, C group, A+C group, ACYW135 group abroad at present, proved in inoculation crowd very safely with effective.But, the capsular polysaccharide on B group meningitis cocci surface may produce cross reaction containing similar structure in mammal and human embryo tissue and ganglioside, so the capsular polysaccharide of B group meningitis is not suitable for the research and development of traditional meningococcal polysaccharide vaccine or GL-PP combined vaccine, find the key that suitable non-k antigen is exploitation B group meningitis cocci vaccine.
Mankind H binding factor is a kind of antigen protein that all Neisseria meningitidis bacterial strains are all expressed, but the HBP aminoacid sequence of expressing due to different strains is different, thus cross-immunoreactivity difference to some extent, therefore can be divided into HBP V1, HBP V2 according to HBP difference, HBP V3 tri-makes a variation kind.
Summary of the invention
The object of the present invention is to provide a kind of B group's epidemic cerebrospinal meningitis coccus recombinant protein vaccine.
Another object of the present invention is to provide the method preparing above-mentioned vaccine.
B group's epidemic cerebrospinal meningitis coccus recombinant protein vaccine of the present invention contains the recombiant protein of mankind H factor bindin three variants, is respectively rHBP V1, rHBP V2, rHBP V3.The nucleotide sequence of coding rHBP V1, rHBP V2, rHBP V3 recombiant protein is respectively as shown in SEQ ID NO.1-3.
RHBP V1 recombiant protein of the present invention prepares by the following method: synthesis rHBP V1 genetic fragment, nucleotide sequence is as shown in SEQ ID NO.1, and hold interpolation Bgl II, Xho I restriction enzyme site respectively at the 5 ' end and 3 ' of this gene, be connected with expression vector, import host cell, screening obtains the engineered strain MH-rHBP V1 of high expression rHBP V1, and deposit number is CGMCC NO.10718; This project strain fermentation is cultivated, abduction delivering recombiant protein, purification, filtration sterilization;
RHBP V2 recombiant protein of the present invention prepares by the following method: synthesis rHBP V2 genetic fragment, nucleotide sequence is as shown in SEQ ID NO.2, and hold interpolation Hind III, Xho I restriction enzyme site respectively at the 5 ' end and 3 ' of this gene, be connected with expression vector, import host cell, screening obtains the engineered strain MH-rHBP V2 of high expression rHBP V2, and deposit number is CGMCC NO.10719; This project strain fermentation is cultivated, abduction delivering recombiant protein, purification, filtration sterilization;
RHBP V3 recombiant protein of the present invention prepares by the following method: synthesis rHBP V3 genetic fragment, nucleotide sequence is as shown in SEQ ID NO.1, and hold interpolation Nde I, Xho I restriction enzyme site respectively at the 5 ' end and 3 ' of this gene, be connected with expression vector, import host cell, screening obtains the engineered strain MH-rHBP V3 of high expression rHBP V3, and deposit number is CGMCC NO.10720; This project strain fermentation is cultivated, abduction delivering recombiant protein, purification, filtration sterilization.
The invention provides the application respectively in the medicine of the disease that preparation prevents or the popular meningoencephalitis for the treatment of B group brings out of above-mentioned rHBP V1, rHBP V2, rHBP V3 recombiant protein.
In embodiments of the invention, when preparing above-mentioned three kinds of engineering strains, expression vector used is pET-22b, 3 engineered strains called after MH-rHBP V1, MH-rHBP V2, MH-rHBP V3 respectively expressing recombiant proteins.
MH-rHBP V1 bacterial strain, its deposit number is CGMCC NO.10718, this bacterial strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 15th, 2015 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), Classification And Nomenclature is colon bacillus Escherichia coli.
MH-rHBP V2 bacterial strain, its deposit number is CGMCC NO.10719, this bacterial strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 15th, 2015 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), Classification And Nomenclature is colon bacillus Escherichia coli.
MH-rHBP V3 bacterial strain, its deposit number is CGMCC NO.10720, this bacterial strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 15th, 2015 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), Classification And Nomenclature is colon bacillus Escherichia coli.
Above-mentioned 3 fermentation culture methods of engineered strain of expressing recombiant proteins are: being inoculated in respectively by engineered strain containing final concentration is the LB fluid medium of 50 μ g/mL ampicillin, 37 DEG C of overnight incubation prepare seed liquor, seed liquor moves in the fluid medium being applicable to Escherichia coli Growth ferments: 37 DEG C, pH7.0-7.2,200r/min;
The method of abduction delivering recombiant protein is: when bacterial growth is to OD
600when=15, add the IPTG induction that final concentration is 0.1mmol/L, inducing temperature is 30 DEG C, after continuing cultivation 6 ~ 8h, stop cultivating, 8000rpm, 4 DEG C of continuous centrifugal 20min, collect thalline, with pH7.0,20mmol/L Tris-HCl buffer solution thalline 2 times, after homogenate fragmentation, 12000g, 4 DEG C of centrifugal 30min, discard precipitation.
In vaccine of the present invention, rHBP V1, rHBP V2, the content of rHBP V3 in vaccine are 22.5-27.5 μ g/ml, regulate pH to 7.0-7.4.
Preferably, in vaccine of the present invention, rHBP V1, rHBP V2, the content of rHBP V3 in vaccine are 25.0 μ g/ml, regulate pH to 7.2.
Vaccine of the present invention also containing adjuvant, described adjuvant be selected from aluminum phosphate, aluminium hydroxide and MF59 one or more; Preferably phosphoric acid aluminum.
In vaccine of the present invention, aluminum content is 0.5-1.5mg/ml vaccine.
The present invention also provides a kind of method preparing B group's epidemic cerebrospinal meningitis coccus recombinant protein vaccine, comprises the following steps:
(1) rHBP V1, rHBP V2, rHBP V3 recombiant protein is prepared; The nucleotide sequence of described recombiant protein is respectively as shown in SEQ ID NO.1-3;
The preparation method of rHBP V1, rHBP V2, rHBP V3 recombiant protein is: rHBP V1, rHBP V2, rHBP V3 gene are connected with carrier respectively, transform, picking identifies correct positive colony, proceed to competent cell, picking monoclonal, inoculation medium, adds IPTG induction and selects the highest bacterial strain of expression to preserve as engineering bacterial strain; The bacterial strain that described expression is the highest is respectively: the MH-rHBP V2 that the MH-rHBP V1 that deposit number is CGMCC NO.10718, deposit number are CGMCC NO.10719, deposit number are the MH-rHBP V3 of CGMCC NO.10720;
(2) 3 the recombiant protein high expressed bacterial strains obtained are fermented amplification culture respectively, abduction delivering recombiant protein; Purifying protein, filtration sterilization;
(3) by 3 kinds of recombiant protein rHBP V1, rHBP V2, rHBP V3 quantitatively after, adsorb with adjuvant respectively, after ultracentrifugation removal endotoxin, three is mixed obtained B group's epidemic cerebrospinal meningitis coccus recombinant protein vaccine.
The carrier of step (1) is pET-22b.
The fermentation culture method of bacterial strain is in step (2): being first inoculated in three strain strains of the present invention containing final concentration is respectively the fresh LB fluid medium of 50 μ g/mL ampicillin, 37 DEG C of overnight incubation prepare seed liquor, for fermentation tank inoculation, fermentation culture can select the culture medium of suitable Escherichia coli Growth; Ferment control parameter setting: 37 DEG C, pH7.0-7.2,200 revs/min.
In step (2), the method for abduction delivering recombiant protein, purification, filtration is: when bacterial growth is to OD
600when=15, add the IPTG induction that final concentration is 0.1mmol/L, inducing temperature is 30 DEG C, after continuing cultivation 6 ~ 8h, stop cultivating, 8000rpm, 4 DEG C of continuous centrifugal 20min, collect thalline, with pH7.0,20mmol/L Tris-HCl buffer solution thalline 2 times, after homogenate fragmentation, 12000g, 4 DEG C of centrifugal 30min, discard precipitation, ion chromatography purification, detects chromatographic peak at a wavelength of 280 nm, collects target peak, obtain rHBP V1 solution, rHBP V1 solution after purification carries out aseptic filtration, quantitatively, subpackage, in 2 ~ 8 DEG C of preservations.
In step (3), adjuvant is aluminium adjuvant, and after three's mixing, aluminum content is 0.5-1.5mg/ml vaccine.
Three mixes rear rHBP V1, rHBP V2, the content of rHBP V3 in vaccine is 22.5-27.5 μ g/ml, regulates pH to 7.0-7.4.Preferably, after mixing, rHBP V1, rHBP V2, the content of rHBP V3 in vaccine are 25.0 μ g/ml, regulate pH to 7.2.
The capsular polysaccharide that the present invention is directed to B group meningitis is not suitable for the shortcoming of the research and development of traditional meningococcal polysaccharide vaccine or GL-PP combined vaccine, utilize technique for gene engineering to express the recombinant protein antigen of people's H factor bindin three variants, prepare a kind of B group's fluidity meningococcus vaccine.The each component of this vaccine is coordinated to supplement mutually; antigenicity is strong; there is good immunogenicity, safety and biologic activity; body can be induced to produce the neutralizing antibody of high titre; and there is bactericidal activity, protection is strong, and vaccine is through lumbar injection immune mouse; mice body can be excited to produce anti-B group's epidemic cerebrospinal meningitis specific antibody, the serum bactericidal antibody of complement-mediated can be made to raise more than 8 times.
Accompanying drawing explanation
Fig. 1 is that after rHBP purification, SDS-PAGE detection figure, Figure 1A figure are rHBP V1SDS-PAGE detection figure, and in figure, 1-3 is the rHBP V1 of purification, and 4 is the foreign protein removed, and 5 is Marker; Figure 1B figure is rHBP V2SDS-PAGE detection figure, and in figure, 1 is the rHBP V2 of purification, and 2 is Marker; Fig. 1 C figure is that rHBP V3SDS-PAGE detects figure, and in figure, 1-3 is the rHBP V3 of purification, and 4 is Marker.
Detailed description of the invention
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, the condition as described in " molecular cloning: laboratory manual " (New York:Cold Spring Harbor Laboratory Press, 1989) is carried out.
Embodiment 1 B group epidemic cerebrospinal meningitis coccus recombinant protein vaccine each component recombination protein gene engineered strain builds
1, the structure of rHBP V1 engineering bacteria
According to the rHBP V1 encoding gene (GenBank accession number: AE002098.2) that NCBI announces, carry out sequence optimisation (SEQ ID No.1) according to e. coli codon Preference, and add Bgl II, Xho I restriction enzyme site respectively in Sequences upstream, downstream, by the rHBP V1 genetic fragment of synthesis, be connected with the expression vector pET-22b of identical nucleic acid endonuclease digestion, import host cell E.coli BL21 (DE3), the engineered strain (expression: 56.0mg/L) of high expression rHBP V1 is obtained through screening, called after: MH-rHBP V1 bacterial strain, its deposit number is CGMCC NO.10718, this bacterial strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 15th, 2015 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), Classification And Nomenclature is colon bacillus Escherichia coli.According to the requirement of " biological product production calibrating bacterium kind rule of management ", carry out the foundation in rHBP V1 expression strain three-class strain storehouse.
2, the structure of rHBP V2 engineering bacteria
According to the rHBP V2 encoding gene (GenBank accession number: DQ523568.1) that NCBI announces, carry out sequence optimisation (SEQ ID No.2) according to e. coli codon Preference, and add Hind III, Xho I restriction enzyme site respectively in Sequences upstream, downstream, by the rHBP V2 of synthesis, with use Hind III, the expression vector pET-22b of Xho I Cobra venom endonuclease enzyme action connects, import host cell E.coli BL21 (DE3), the engineered strain of high expression (73.1mg/L) rHBP V2 is obtained through screening, called after: MH-rHBP V2 bacterial strain, its deposit number is CGMCC NO.10719, this bacterial strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 15th, 2015 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), Classification And Nomenclature is colon bacillus Escherichia coli.According to the requirement of " biological product production calibrating bacterium kind rule of management ", carry out the foundation in rHBP V2 expression strain three-class strain storehouse.
3, the structure of rHBP V3 engineering bacteria
According to the rHBP V3 encoding gene (GenBank accession number: DQ523569.1) that NCBI announces, carry out sequence optimisation (SEQ ID No.3) according to e. coli codon Preference, and add Nde I, Xho I restriction enzyme site respectively in Sequences upstream, downstream, by the rHBP V3 of synthesis, with use Nde I, the expression vector pET-22b of Xho I Cobra venom endonuclease enzyme action connects, import host cell E.coli BL21 (DE3), the engineered strain of high expression (68.6mg/L) rHBP V3 is obtained through screening, called after: MH-rHBP V3 bacterial strain, its deposit number is CGMCC NO.10720, this bacterial strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 15th, 2015 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), Classification And Nomenclature is colon bacillus Escherichia coli.According to the requirement of " biological product production calibrating bacterium kind rule of management ", carry out the foundation in rHBP V3 expression strain three-class strain storehouse.
The acquisition of embodiment 2 rHBP V1, rHBP V2, rHBP V3 recombiant protein
The acquisition of rHBP tri-variant rHBP V1, rHBP V2, rHBP V3, process is divided into: seed liquor preparation, fermentation, collected by centrifugation thalline, bacterial cell disruption, filtration sterilization and cryopreservation.Be below the preparation method of rHBP V1, the preparation method of rHBP V2, rHBP V3 is with reference to rHBP V1.
RHBP V1 working seed lots strain (the MH-rHBP V1 bacterial strain first embodiment 1 obtained, its deposit number is CGMCC NO.10718) to be inoculated in respectively containing final concentration be the fresh LB fluid medium of 50 μ g/mL ampicillin, 37 DEG C of overnight incubation prepare seed liquor, for fermentation tank inoculation, fermentation culture can select the culture medium of suitable Escherichia coli Growth.Fermentation parameter: 37 DEG C, pH7.0-7.2,200 turns; When bacterial growth is to OD
600when=15, add the IPTG induction that final concentration is 0.1mmol/L, inducing temperature is 30 DEG C, after continuing cultivation 6 ~ 8h, stop cultivating, 8000rpm, 4 DEG C of continuous centrifugal 20min, collect thalline, with pH7.0,20mmol/L Tris-HCl buffer solution thalline 2 times, after homogenate fragmentation, 12000g, 4 DEG C of centrifugal 30min, discard precipitation, ion chromatography purification, detects chromatographic peak at a wavelength of 280 nm, collect target peak, obtain rHBP V1 solution.RHBP V1 solution after purification carries out aseptic filtration, quantitatively, is sub-packed in large bottle, in 2 ~ 8 DEG C of preservations.
Preparation and B group's epidemic cerebrospinal meningitis coccus recombinant protein vaccine of embodiment 3 antigen and aluminum hydroxide adjuvant adsorbed product are prepared
Aluminum hydroxide adjuvant preparation method is: in PBS, slowly adds 10%KAl (SO
4)
2, magnetic stirring apparatus, is as the criterion not form whirlpool, mixing.Slowly 0.5mol/L NaOH is dripped, until pH reaches 7.0 stoppings in mixed liquor.4 DEG C, after leaving standstill 24h, change liquid with 0.85% physiological sodium chloride solution, and standardize solution is to initial volume.Change liquid altogether four times, every minor tick 8h.Change liquid to complete at every turn, stir the rearmounted 4 DEG C of refrigerators of 30min and place.Change liquid NaOH for the last time and adjust pH to 6.0-7.0 standardize solution, use front HIGH PRESSURE TREATMENT.
1, the preparation of rHBP V1 and aluminum hydroxide adjuvant adsorbed product
The rHBP V1 albumen of the aseptic filtration that Example 2 is obtained, regulates pH to 5.5-6.5, adds isopyknic aluminium hydroxide, obtain final concentration be 1.5mg/mL aluminium adjuvant mixing material, mixing, room temperature absorption 1h, 4 DEG C of preservations.
2, the preparation of rHBP V2 and aluminum hydroxide adjuvant adsorbed product
The rHBP V2 albumen of the aseptic filtration that Example 2 is obtained, regulates pH to 6.0-7.0, adds isopyknic aluminium hydroxide, obtain the aluminium adjuvant mixing material that final concentration is 1.5mg/mL, mixing, room temperature absorption 1h, 4 DEG C of preservations.
3, the preparation of rHBP V3 and aluminum hydroxide adjuvant adsorbed product
The rHBP V3 albumen of the aseptic filtration that Example 2 is obtained, regulates pH to 6.5-7.0, adds isopyknic aluminium hydroxide, obtain the aluminium adjuvant mixing a liquid that final concentration is 1.5mg/mL, mixing, room temperature absorption 1h, 4 DEG C of preservations.
3 kinds of rHBP variant protein of above-mentioned preparation and the adsorbed product of aluminum hydroxide adjuvant is diluted with 5mmol/L phosphate buffer (pH7.2), then by 3 kinds of adsorbed product equal-volume mixing, make the content of three of rHBP variants V1, V2, V3 be 25 μ g/ml, regulate pH to 7.2.
Preparation and B group's epidemic cerebrospinal meningitis coccus recombinant protein vaccine of embodiment 4 antigen and Aluminium phosphate adjuvant adsorbed product are prepared
Aluminium phosphate adjuvant preparation is carried out, pH regulator to 6.5 according to patent (CN 101730660 A).
1, the preparation of rHBP V1 and Aluminium phosphate adjuvant adsorbed product
The rHBP V1 albumen of the aseptic filtration that Example 2 is obtained, regulates pH to 5.5-6.5, adds isopyknic aluminium hydroxide, obtain final concentration be 1mg/mL aluminium adjuvant mixing material, mixing, room temperature absorption 1h, 4 DEG C of preservations.
2, the preparation of rHBP V2 and Aluminium phosphate adjuvant adsorbed product
The rHBP V2 albumen of the aseptic filtration that Example 2 is obtained, regulates pH to 6.0-7.0, adds isopyknic aluminium hydroxide, obtain the aluminium adjuvant mixing material that final concentration is 1mg/mL, mixing, room temperature absorption 1h, 4 DEG C of preservations.
3, the preparation of rHBP V3 and Aluminium phosphate adjuvant adsorbed product
The rHBP V3 albumen of the aseptic filtration that Example 2 is obtained, regulates pH to 6.5-7.0, adds isopyknic aluminium hydroxide, obtain the aluminium adjuvant mixing material that final concentration is 1mg/mL, mixing, room temperature absorption 1h, 4 DEG C of preservations.
The absorption system of rHBP and Aluminium phosphate adjuvant in 3 of above-mentioned preparation is diluted with 5mmol/L phosphate buffer (pH7.2), then by 3 kinds of adsorbed product equal-volume mixing, make finished product component and content to be: the content of three variants V1, V2, V3 of rHBP is 25.0 μ g/ml, regulate pH to 7.2.
The bactericidal assay of embodiment 5 B group epidemic cerebrospinal meningitis coccus recombinant protein vaccine
Be 16-18g female BAl BIc/c mice random packet by body weight, often organize 10, carry out immunity with B group popular meningoencephalitis coccus recombinant protein vaccine prepared by embodiment 3 and embodiment 4 respectively, establish saline control group simultaneously.Every mouse peritoneal injection 0.5ml, containing each 25 μ g of rHBP V1, V2, V3, in each immunity in the 0th day, the 21st day, the 35th day once, eye socket blood sampling in the 14th day after final immunization, separation of serum, aseptic subpackagedly carries out the coherent detections such as sterilizing power.
1, TTC liquid preparation: make 0.5% (W/V) TTC solution with sterile purified water, heating for dissolving, is placed in brown bottle, and 2-8 DEG C saves backup.
2, indicate collecting cells, B group's epidemic cerebrospinal meningitis coccus 341215 is inoculated in blood agar plate, 5%CO
2, 37 DEG C cultivate 16-18h after, wash lower lawn with nutrient broth, measure OD value with spectrophotometer in 600nm, adjustment OD value to 0.1 is stock solution, is 1:10000 and dilutes and (be equivalent to 10
4cfu/mL) use afterwards.
3, complement-instruction collecting cells: with sterilizing PBS (pH7.4) or normal saline, rabbit complement is done 1:20 dilution, then add the indicator bacteria that final concentration is 1% (v/v).
4, microtitration plate: 4 × 10 hole polystyrene plastic plates, its carrying out washing treatment requires same ELISA method.With micro-liquid feeding dropper, PBS or normal saline are dripped 1 (0.025mL) in every hole of titer plate respectively with vertical direction, detected inactivated serum is added one (0.025mL) in the first hole that each is arranged, every block plate can do 4 parts of specimen again.
5, add complement-indicator bacteria suspension: from often to ranked first hole start to do 2 times be diluted to the tenth hole after, every hole adds complement-indicator bacteria suspension 1, adds a cover, microoscillator shakes 90s, put wet pans, cultivates 30min in 37 DEG C of water baths.
6, TTC nutrient broth is added: every hole adds containing 0.01%TTC nutrient broth 0.15mL, after mixing, continues to put in wet pans, cultivates 4h in 37 DEG C of water baths.
7, observed result: Sptting plate is placed on observed result on blank sheet of paper, with in the hole of the most high dilution of serum without color change person for detecting the titre of killing B group's epidemic cerebrospinal meningitis coccus antibody of specimen.
Table 1 TTC method detects the bactericidin that B group popular meningoencephalitis coccus recombinant protein vaccine excites
The bactericidin titre of complement-mediated improves 8 times or higher and has clinical meaning, sterilizing power result (table 1) display of B group popular meningoencephalitis coccus recombinant protein vaccine, the antigen of two kinds of aluminium adjuvant absorption can excite the generation of mice antibody against toxin, and the bactericidin titre that wherein popular meningoencephalitis coccus recombinant protein vaccine (Aluminium phosphate adjuvant) of B group produces raises especially obvious.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. B group's epidemic cerebrospinal meningitis coccus recombinant protein vaccine, is characterized in that, the recombiant protein containing mankind H factor bindin three variants, is respectively rHBP V1, rHBP V2, rHBP V3.
2. vaccine as claimed in claim 1, is characterized in that, the nucleotide sequence of coding rHBP V1, rHBP V2, rHBP V3 recombiant protein is respectively as shown in SEQ ID NO.1-3.
3. vaccine as claimed in claim 1, is characterized in that,
RHBP V1 recombiant protein prepares by the following method: synthesis rHBP V1 genetic fragment, nucleotide sequence is as shown in SEQ ID NO.1, and hold interpolation Bgl II, Xho I restriction enzyme site respectively at the 5 ' end and 3 ' of this gene, be connected with expression vector, import host cell, screening obtains the engineered strain MH-rHBP V1 of high expression rHBP V1, and deposit number is CGMCC NO.10718; This project strain fermentation is cultivated, abduction delivering recombiant protein, purification, filtration sterilization;
RHBP V2 recombiant protein prepares by the following method: synthesis rHBP V2 genetic fragment, nucleotide sequence is as shown in SEQ ID NO.2, and hold interpolation Hind III, Xho I restriction enzyme site respectively at the 5 ' end and 3 ' of this gene, be connected with expression vector, import host cell, screening obtains the engineered strain MH-rHBP V2 of high expression rHBP V2, and deposit number is CGMCC NO.10719; This project strain fermentation is cultivated, abduction delivering recombiant protein, purification, filtration sterilization;
RHBP V3 recombiant protein prepares by the following method: synthesis rHBP V3 genetic fragment, nucleotide sequence is as shown in SEQ ID NO.1, and hold interpolation Nde I, Xho I restriction enzyme site respectively at the 5 ' end and 3 ' of this gene, be connected with expression vector, import host cell, screening obtains the engineered strain MH-rHBP V3 of high expression rHBP V3, and deposit number is CGMCC NO.10720; This project strain fermentation is cultivated, abduction delivering recombiant protein, purification, filtration sterilization.
4. vaccine as claimed in claim 1, it is characterized in that, rHBP V1, rHBP V2, the content of rHBP V3 in vaccine are 22.5-27.5 μ g/ml, regulate pH to 7.0-7.4.
5. vaccine as claimed in claim 4, it is characterized in that, rHBP V1, rHBP V2, the content of rHBP V3 in vaccine are 25.0 μ g/ml, regulate pH to 7.2.
6. vaccine as claimed in claim 1, is characterized in that, also containing adjuvant, described adjuvant be selected from aluminum phosphate, aluminium hydroxide and MF59 one or more.
7. vaccine as claimed in claim 6, it is characterized in that, aluminum content is 0.5-1.5mg/ml vaccine.
8. prepare a method for the arbitrary described B group's epidemic cerebrospinal meningitis coccus recombinant protein vaccine of claim 1-6, it is characterized in that, comprise the following steps:
(1) rHBP V1, rHBP V2, rHBP V3 recombiant protein is prepared; The nucleotide sequence of described recombiant protein is respectively as shown in SEQ ID NO.1-3;
The preparation method of rHBP V1, rHBP V2, rHBP V3 recombiant protein is: rHBP V1, rHBP V2, rHBP V3 gene are connected with carrier respectively, transform, picking identifies correct positive colony, proceed to competent cell, picking monoclonal, inoculation medium, adds IPTG induction and selects the highest bacterial strain of expression to preserve as engineering bacterial strain; The bacterial strain that described expression is the highest is respectively: MH-rHBP V1 deposit number is CGMCC NO.10718, MH-rHBP V2 deposit number is CGMCC NO.10719, MH-rHBP V3 deposit number is CGMCC NO.10720;
(2) 3 the recombiant protein high expressed bacterial strains obtained are fermented amplification culture respectively, abduction delivering recombiant protein; Purifying protein, filtration sterilization;
(3) by 3 kinds of recombiant protein rHBP V1, rHBP V2, rHBP V3 quantitatively after, adsorb with adjuvant respectively, three mixes after removing endotoxin by ultracentrifugation, obtains B group's epidemic cerebrospinal meningitis coccus recombinant protein vaccine.
9. method as claimed in claim 8, is characterized in that, in step (3), adjuvant is aluminium adjuvant, and after three's mixing, aluminum content is 0.5-1.5mg/ml vaccine.
10. method as claimed in claim 8 or 9, it is characterized in that, rHBP V1, rHBP V2, the content of rHBP V3 in vaccine are 22.5-27.5 μ g/ml, regulate pH to 7.0-7.4.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106215183A (en) * | 2016-07-21 | 2016-12-14 | 北京智飞绿竹生物制药有限公司 | A kind of ABC group meningitis cocci combined vaccine and preparation method thereof |
| CN107823638A (en) * | 2017-11-05 | 2018-03-23 | 北京智飞绿竹生物制药有限公司 | A kind of B group meningitis coccis restructuring chimeric protein vaccine and preparation method thereof |
| CN110818779A (en) * | 2019-12-20 | 2020-02-21 | 北京民海生物科技有限公司 | Group B epidemic encephalitis fHbp-V2 recombinant protein and preparation method thereof, vaccine composition and application |
| CN110903360A (en) * | 2019-12-20 | 2020-03-24 | 北京民海生物科技有限公司 | Group B epidemic encephalitis fHbp-V3 recombinant protein and preparation method thereof, vaccine composition and application |
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| CN104072590A (en) * | 2014-06-24 | 2014-10-01 | 上海生物制品研究所有限责任公司 | Meningococcus antigen combination and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106215183A (en) * | 2016-07-21 | 2016-12-14 | 北京智飞绿竹生物制药有限公司 | A kind of ABC group meningitis cocci combined vaccine and preparation method thereof |
| CN107823638A (en) * | 2017-11-05 | 2018-03-23 | 北京智飞绿竹生物制药有限公司 | A kind of B group meningitis coccis restructuring chimeric protein vaccine and preparation method thereof |
| CN107823638B (en) * | 2017-11-05 | 2020-01-03 | 北京智飞绿竹生物制药有限公司 | Group B meningococcus recombinant chimeric protein vaccine and preparation method thereof |
| CN110818779A (en) * | 2019-12-20 | 2020-02-21 | 北京民海生物科技有限公司 | Group B epidemic encephalitis fHbp-V2 recombinant protein and preparation method thereof, vaccine composition and application |
| CN110903360A (en) * | 2019-12-20 | 2020-03-24 | 北京民海生物科技有限公司 | Group B epidemic encephalitis fHbp-V3 recombinant protein and preparation method thereof, vaccine composition and application |
| CN110818779B (en) * | 2019-12-20 | 2021-10-08 | 北京民海生物科技有限公司 | Group B epidemic encephalitis fHbp-V2 recombinant protein and preparation method thereof, vaccine composition and application |
| CN110903360B (en) * | 2019-12-20 | 2021-10-08 | 北京民海生物科技有限公司 | Group B epidemic encephalitis fHbp-V3 recombinant protein and preparation method thereof, vaccine composition and application |
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