CN104888209B - A kind of B groups of epidemic meningitises coccus recombinant protein vaccine and preparation method thereof - Google Patents
A kind of B groups of epidemic meningitises coccus recombinant protein vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention provides a kind of B groups of epidemic meningitises coccus recombinant protein vaccine and preparation method thereof, belong to field of biological product.Three variant recombinant proteins of vaccine H containing someone factor bindins of the present invention.Present invention also offers the preparation method of B groups of epidemic meningitis coccus recombinant protein vaccines, including：The mankind H factor bindin V1, V2, V3 recombinant expression carrier build, express bacterial strain screening identification, fermented and cultured, the induced expression of recombinant protein antigen, the purifying of recombinant protein antigen and Protein Detection, according to the concentration of above-mentioned 3 kinds of antigenic components, it is made after quantitative mixing with reference to aluminium adjuvant.The popular meningoencephalitis coccus recombinant protein vaccine of B groups that the present invention is provided can excite mouse body to produce anti-B groups of epidemic meningitis specific antibodies, the serum bactericidal antibody of complement-mediated can be made to raise more than 8 times through abdominal cavity injecting immune mouse.
Description
Technical Field
The invention relates to the field of biological products, in particular to a group B epidemic meningococcus recombinant protein vaccine and a preparation method thereof.
Background
Neisseria meningitidis is the main pathogenic bacterium of bacterial meningitis and sepsis, and according to the difference of structure and antigenicity of capsular polysaccharide on the surface of meningococcus thallus, meningococcus is divided into 13 serogroups (Serrogoup), wherein 5 groups (A, B, C, W135 and Y groups) are the main pathogenic flora. The incidence of meningitis worldwide by meningococci is 30-50 million per year, with about 50% of those infected being predominantly by group B meningococci. In recent years, drug-resistant strains have emerged in succession around the world due to abuse of antibiotics, which has added greater difficulty to the treatment of diseases.
The safe and effective vaccination is generally considered as an ideal measure for preventing and controlling epidemic encephalitis, cross protection is not provided among all pathogenic serotypes of meningococcus, the univalent vaccination cannot effectively prevent infection of other serotype strains, and various univalent, bivalent and tetravalent epidemic encephalitis polysaccharide and polysaccharide protein combined vaccines such as A group, C group, A + C group and ACYW135 group are sold on the market abroad at present and are proved to be very safe and effective in vaccinated people. However, capsular polysaccharide on the surface of group B meningococcus may have similar structures in embryonic tissues and ganglioside of mammals and humans, and thus, the capsular polysaccharide of group B meningococcus is not suitable for research and development of traditional meningococcus polysaccharide vaccines or polysaccharide protein conjugate vaccines, and searching for suitable non-capsular antigen is the key for developing group B meningococcus vaccines.
The human H-binding factor is an antigen protein expressed by all strains of Neisseria meningitidis, but has different cross-immunoreactivity due to different HBP amino acid sequences expressed by different strains, so that the human H-binding factor can be divided into three variants of HBPV1, HBP V2 and HBP V3 according to the HBP difference.
Disclosure of Invention
The invention aims to provide a group B epidemic meningococcus recombinant protein vaccine.
It is another object of the present invention to provide a method for preparing the above vaccine.
The B group epidemic meningococcus recombinant protein vaccine contains recombinant proteins of three variants of human H factor binding protein, namely rHBP V1, rHBP V2 and rHBP V3. The nucleotide sequences of the recombinant proteins for coding rHBP V1, rHBP V2 and rHBP V3 are respectively shown in SEQ ID NO. 1-3.
The rHBP V1 recombinant protein is prepared by the following method: synthesizing rHBP V1 gene fragment, wherein the nucleotide sequence is shown as SEQ ID NO.1, adding Bgl II and Xho I enzyme cutting sites at the 5 'end and the 3' end of the gene respectively, connecting with an expression vector, introducing into host cells, and screening to obtain an engineering strain MH-rHBP V1 for efficiently expressing rHBP V1, wherein the preservation number is CGMCC NO. 10718; fermenting and culturing the engineering strain, inducing to express recombinant protein, purifying, filtering and sterilizing;
the rHBP V2 recombinant protein is prepared by the following method: synthesizing rHBP V2 gene fragment, wherein the nucleotide sequence is shown as SEQ ID NO.2, adding Hind III and Xho I enzyme cutting sites at the 5 'end and the 3' end of the gene respectively, connecting with an expression vector, introducing into host cells, and screening to obtain an engineering strain MH-rHBP V2 for efficiently expressing rHBP V2, wherein the preservation number is CGMCC NO. 10719; fermenting and culturing the engineering strain, inducing to express recombinant protein, purifying, filtering and sterilizing;
the rHBP V3 recombinant protein is prepared by the following method: synthesizing rHBP V3 gene fragment, wherein the nucleotide sequence is shown as SEQ ID NO.1, Nde I enzyme cutting sites and Xho I enzyme cutting sites are respectively added at the 5 'end and the 3' end of the gene, the enzyme cutting sites are connected with an expression vector, the enzyme cutting sites are introduced into a host cell, and an engineering strain MH-rHBP V3 for efficiently expressing rHBP V3 is obtained by screening, wherein the preservation number is CGMCC NO. 10720; fermenting and culturing the engineering strain, inducing to express recombinant protein, purifying, filtering and sterilizing.
The invention provides application of the rHBP V1, rHBP V2 and rHBP V3 recombinant proteins in preparation of medicines for preventing or treating diseases induced by group B epidemic meningoencephalitis.
In the embodiment of the invention, expression vectors used for preparing the three genetic engineering strains are pET-22b, and 3 engineering strains for expressing recombinant proteins are respectively named MH-rHBP V1, MH-rHBP V2 and MH-rHBP V3.
MH-rHBP V1 strain with the preservation number of CGMCC NO.10718, which has been preserved in China general microbiological culture Collection center (CGMCC for short, address: Beijing city, Chaoyang district, Beicheng West Lu No.1 institute of microbiology, China academy of sciences, zip code 100101) 4.15 days 2015, and is named as Escherichia coli by classification.
MH-rHBP V2 strain with the preservation number of CGMCC NO.10719, which has been preserved in the China general microbiological culture Collection center (CGMCC for short, the address: Beijing city, Chaoyang district, Beichen Xilu No.1 institute of microbiology, China academy of sciences, zip code 100101) 4.15 days 2015, and is named as Escherichia coli by classification.
MH-rHBP V3 strain with the preservation number of CGMCC NO.10720, which has been preserved in China general microbiological culture Collection center (CGMCC for short, address: Beijing city, Chaoyang district, Beicheng West Lu No.1 institute of microbiology, China academy of sciences, zip code 100101) 4.15 days 2015, and is named as Escherichia coli by classification.
The fermentation culture method of the 3 engineering strains for expressing the recombinant protein comprises the following steps: respectively inoculating the engineering strains into LB liquid culture medium containing ampicillin with the final concentration of 50 mug/mL, culturing overnight at 37 ℃ to prepare seed liquid, transferring the seed liquid into liquid culture medium suitable for growth of escherichia coli to ferment: at 37 ℃, the pH value is 7.0-7.2, and the speed is 200 r/min;
the method for inducing and expressing the recombinant protein comprises the following steps: when the bacteria grew to OD600When the concentration is 15 ℃, adding IPTG with the final concentration of 0.1mmol/L for induction, keeping the induction temperature at 30 ℃, continuing to culture for 6-8 h, stopping culturing, continuously centrifuging at 8000rpm and 4 ℃ for 20min, collecting thalli, washing the thalli for 2 times by using Tris-HCl buffer solution with the pH value of 7.0 and 20mmol/L, homogenizing and crushing, then 12000g, centrifuging at 4 ℃ for 30min, and discarding the precipitate.
In the vaccine, the contents of rHBP V1, rHBP V2 and rHBP V3 in the vaccine are 22.5-27.5 mu g/ml, and the pH is adjusted to 7.0-7.4.
Preferably, in the vaccine of the invention, the content of rHBP V1, rHBP V2 and rHBP V3 in the vaccine is 25.0 μ g/ml, and the pH is adjusted to 7.2.
The vaccine also comprises an adjuvant, wherein the adjuvant is selected from one or more of aluminum phosphate, aluminum hydroxide and MF 59; aluminum phosphate is preferred.
In the vaccine of the invention, the aluminum content is 0.5-1.5mg/ml vaccine.
The invention also provides a method for preparing the B group epidemic meningococcus recombinant protein vaccine, which comprises the following steps:
(1) preparing rHBP V1, rHBP V2 and rHBP V3 recombinant proteins; the nucleotide sequences of the recombinant proteins are respectively shown in SEQ ID NO. 1-3;
the preparation method of rHBP V1, rHBP V2 and rHBP V3 recombinant proteins comprises the following steps: respectively connecting rHBP V1, rHBP V2 and rHBP V3 genes with a vector, transforming, selecting and identifying a correct positive clone, transferring the positive clone into a competent cell, selecting a monoclonal, inoculating a culture medium, and adding IPTG (isopropyl thiogalactoside) to induce and select a strain with the highest expression quantity to be used as an engineering strain for storage; the strains with the highest expression amount are respectively as follows: MH-rHBP V1 with the preservation number of CGMCC NO.10718, MH-rHBP V2 with the preservation number of CGMCC NO.10719 and MH-rHBP V3 with the preservation number of CGMCC NO. 10720;
(2) respectively fermenting and expanding the obtained 3 recombinant protein high-expression strains for culture, and inducing to express recombinant proteins; purifying protein, filtering and sterilizing;
(3) quantifying 3 recombinant proteins rHBP V1, rHBP V2 and rHBP V3, respectively adsorbing with adjuvant, ultracentrifuging to remove endotoxin, and mixing to obtain group B epidemic meningococcal recombinant protein vaccine.
The vector in the step (1) is pET-22 b.
The fermentation culture method of the strain in the step (2) comprises the following steps: firstly, respectively inoculating the three strains of the invention into fresh LB liquid culture medium containing ampicillin with the final concentration of 50 mug/mL, culturing overnight at 37 ℃ to prepare seed liquid for inoculating in a fermentation tank, and selecting a culture medium suitable for growth of escherichia coli in fermentation culture; setting fermentation control parameters: at 37 ℃ and pH7.0-7.2, 200 rpm.
The method for inducing expression of the recombinant protein, purification and filtration in the step (2) comprises the following steps: when the bacteria grew to OD600When the concentration is 15 ℃, adding IPTG with the final concentration of 0.1mmol/L for induction, keeping the induction temperature at 30 ℃, continuing to culture for 6-8 h, stopping culture, continuously centrifuging at 8000rpm and 4 ℃ for 20min, collecting thalli, washing the thalli for 2 times by using Tris-HCl buffer solution with the pH value of 7.0 and 20mmol/L, homogenizing and crushing, 12000g, centrifuging at 4 ℃ for 30min, discarding the precipitate, purifying by ion chromatography, detecting the chromatographic peak under the wavelength of 280nm, collecting the target peak to obtain rHBP V1 solution, sterilizing and filtering the purified rHBP V1 solution, quantifying, subpackaging, and storing at 2-8 ℃.
The adjuvant in the step (3) is aluminum adjuvant, and after the aluminum adjuvant, the aluminum content of the vaccine is 0.5-1.5 mg/ml.
The rHBP V1, rHBP V2 and rHBP V3 in the vaccine are mixed to reach content of 22.5-27.5 μ g/ml, and pH is regulated to 7.0-7.4. Preferably, the rHBP V1, rHBP V2 and rHBP V3 after mixing are all 25.0 μ g/ml in the vaccine, and the pH is adjusted to 7.2.
Aiming at the defect that capsular polysaccharide of group B meningococcus is not suitable for the development of traditional meningococcus polysaccharide vaccines or polysaccharide protein conjugate vaccines, the invention prepares and obtains the group B epidemic meningococcus vaccine by expressing recombinant protein antigens of three variants of human factor H conjugate protein by using a genetic engineering technology. The vaccine has the advantages of mutually coordinated and supplemented components, strong antigenicity, good immunogenicity, safety and biological activity, capability of inducing an organism to generate a high-titer neutralizing antibody, bactericidal activity and strong protection, and the vaccine can stimulate the organism of a mouse to generate an anti-group B epidemic meningitis specific antibody by injecting the vaccine into an abdominal cavity to immunize the mouse, so that the complement-mediated serum bactericidal antibody can be increased by more than 8 times.
Drawings
FIG. 1 is a SDS-PAGE detection of rHBP after purification, FIG. 1A is a SDS-PAGE detection of rHBP V1, in which 1-3 are purified rHBP V1, 4 are removed hetero-proteins, and 5 are markers; FIG. 1B is a SDS-PAGE of rHBP V2, wherein 1 is purified rHBP V2 and 2 is Marker; FIG. 1C is an SDS-PAGE of rHBP V3, in which 1-3 are purified rHBP V3 and 4 are Marker.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
The experimental methods in the following examples, in which specific conditions are not specified, are generally performed according to conventional conditions, such as "molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).
Example 1 construction of recombinant protein Gene engineering strains for Components of group B epidemic meningococcal recombinant protein vaccine
1. Construction of rHBP V1 engineering bacteria
According to rHBP V1 encoding gene (GenBank accession number: AE002098.2) published by NCBI, sequence optimization (SEQ ID No.1) is carried out according to the codon preference of escherichia coli, and Bgl II enzyme cutting sites and Xho I enzyme cutting sites are respectively added at the upstream and the downstream of the sequence; the synthesized rHBP V1 gene fragment is connected with an expression vector pET-22b cut by the same endonuclease, and is introduced into a host cell E.coli BL21(DE3), and an engineering strain (expression amount: 56.0mg/L) for efficiently expressing rHBP V1 is obtained by screening, and is named as: MH-rHBP V1 strain with the preservation number of CGMCC NO.10718, which has been preserved in China general microbiological culture Collection center (CGMCC for short, address: Beijing city, Chaoyang district, Beicheng West Lu No.1 institute of microbiology, China academy of sciences, zip code 100101) 4.15 days 2015, and is named as Escherichia coli by classification. According to the requirements of the 'bacteria and virus management protocol for biological product production and verification', a rHBP V1 expression strain tertiary strain library is established.
2. Construction of rHBP V2 engineering bacteria
According to the rHBP V2 encoding gene (GenBank accession number: DQ523568.1) published by NCBI, sequence optimization (SEQ ID No.2) is carried out according to the codon preference of escherichia coli, and Hind III and Xho I enzyme cutting sites are respectively added at the upstream and the downstream of the sequence; the synthesized rHBP V2 is connected with an expression vector pET-22b cut by Hind III and Xho I endonuclease, is introduced into a host cell E.coli BL21(DE3), and an engineering strain for efficiently expressing (73.1mg/L) rHBP V2 is obtained by screening and is named as: MH-rHBP V2 strain with the preservation number of CGMCC NO.10719, which has been preserved in the China general microbiological culture Collection center (CGMCC for short, the address: Beijing city, Chaoyang district, Beichen Xilu No.1 institute of microbiology, China academy of sciences, zip code 100101) 4.15 days 2015, and is named as Escherichia coli by classification. According to the requirements of the 'bacteria and virus management protocol for biological product production and verification', a rHBP V2 expression strain tertiary strain library is established.
3. Construction of rHBP V3 engineering bacteria
According to rHBP V3 encoding gene (GenBank accession number: DQ523569.1) published by NCBI, sequence optimization (SEQ ID No.3) is carried out according to the codon preference of escherichia coli, and Nde I enzyme cutting sites and Xho I enzyme cutting sites are respectively added at the upstream and the downstream of the sequence; the synthesized rHBP V3 is connected with an expression vector pET-22b cut by Nde I and Xho I endonucleases, is introduced into a host cell E.coli BL21(DE3), and an engineering strain for efficiently expressing (68.6mg/L) rHBP V3 is obtained by screening and is named as: MH-rHBP V3 strain with the preservation number of CGMCC NO.10720, which has been preserved in China general microbiological culture Collection center (CGMCC for short, address: Beijing city, Chaoyang district, Beicheng West Lu No.1 institute of microbiology, China academy of sciences, zip code 100101) 4.15 days 2015, and is named as Escherichia coli by classification. According to the requirements of the 'bacteria and virus management protocol for biological product production and verification', a rHBP V3 expression strain tertiary strain library is established.
Example 2 production of rHBP V1, rHBP V2, rHBP V3 recombinant proteins
Three variants of rHBP, namely rHBP V1, rHBP V2 and rHBP V3 are obtained, and the process is divided into: preparing seed liquid, fermenting, centrifugally collecting thalli, crushing the thalli, filtering for sterilization and storing at low temperature. The method for obtaining rHBP V1 is described below, and rHBP V2 and rHBPV3 are referred to as rHBP V1.
The batch strain of rHBP V1 working seeds (MH-rHBP V1 strain with preservation number CGMCC NO.10718) obtained in example 1 was inoculated to a fresh LB liquid medium containing ampicillin at a final concentration of 50. mu.g/mL, respectively, and cultured overnight at 37 ℃ to prepare a seed solution for inoculation in a fermenter, and the culture medium suitable for growth of Escherichia coli was selected for fermentation culture. Fermentation parameters: at 37 deg.C, pH7.0-7.2, 200 turns; when the bacteria grew to OD600And (2) when the concentration is 15, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.1mmol/L for induction, keeping the induction temperature at 30 ℃, continuing to culture for 6-8 h, stopping culturing, continuously centrifuging at 8000rpm and 4 ℃ for 20min, collecting thalli, washing the thalli for 2 times by using Tris-HCl buffer solution with the pH value of 7.0 and 20mmol/L, homogenizing and crushing, then 12000g, centrifuging at 4 ℃ for 30min, discarding the precipitate, purifying by ion chromatography, detecting a chromatographic peak at the wavelength of 280nm, and collecting a target peak to obtain rHBP V1 solution. And (3) sterilizing, filtering and quantifying the purified rHBP V1 solution, subpackaging in large bottles, and storing at 2-8 ℃.
Example 3 preparation of antigen and aluminium hydroxide adjuvant adsorption product and preparation of group B epidemic meningococcal recombinant protein vaccine
The preparation method of the aluminum hydroxide adjuvant comprises the following steps: in PBS, 10% KAl (SO) was slowly added4)2And a magnetic stirrer for uniformly mixing the components without forming vortex. To the mixture was slowly added dropwise 0.5mol/L NaOH until the pH reached 7.0. Standing at 4 deg.C for 24 hr, adding 0.85% physiological sodium chlorideThe solution was changed and the volume was adjusted to the initial volume. The solution was changed four times with an interval of 8 h. After each liquid change is finished, stirring for 30min, and placing in a refrigerator at 4 ℃. Adjusting pH to 6.0-7.0 with NaOH to constant volume, and treating under high pressure before use.
1. Preparation of rHBP V1 and aluminum hydroxide adjuvant adsorption product
The sterile filtered rHBP V1 protein prepared in example 2 is taken, the pH is adjusted to 5.5-6.5, equal volume of aluminum hydroxide is added to obtain aluminum adjuvant mixed liquid with the final concentration of 1.5mg/mL, the mixture is uniformly mixed, the mixture is adsorbed for 1h at room temperature, and the mixture is stored at 4 ℃.
2. Preparation of rHBP V2 and aluminum hydroxide adjuvant adsorption product
The sterile filtered rHBP V2 protein prepared in example 2 is taken, the pH is adjusted to 6.0-7.0, equal volume of aluminum hydroxide is added to obtain aluminum adjuvant mixed liquid with the final concentration of 1.5mg/mL, the mixture is uniformly mixed, the mixture is adsorbed for 1h at room temperature, and the mixture is stored at 4 ℃.
3. Preparation of rHBP V3 and aluminum hydroxide adjuvant adsorption product
The sterile filtered rHBP V3 protein prepared in example 2 was taken, pH was adjusted to 6.5-7.0, an equal volume of aluminum hydroxide was added to obtain an aluminum adjuvant mixed liquid a with a final concentration of 1.5mg/mL, mixed well, adsorbed at room temperature for 1h, and stored at 4 ℃.
The adsorption products of 3 kinds of rHBP variant proteins prepared above and aluminum hydroxide adjuvant were diluted with 5mmol/L phosphate buffer (pH7.2), and then the 3 kinds of adsorption products were mixed in equal volumes so that the contents of three variants of rHBP, V1, V2, and V3 were 25. mu.g/ml, and the pH was adjusted to 7.2.
Example 4 preparation of adsorbed product of antigen with aluminum phosphate adjuvant and formulation of group B epidemic meningococcal recombinant protein vaccine
The aluminum phosphate adjuvant preparation was carried out as per patent (CN 101730660 a) with pH adjusted to 6.5.
1. Preparation of rHBP V1 adsorption product with aluminum phosphate adjuvant
The sterile filtered rHBP V1 protein prepared in example 2 is taken, the pH is adjusted to 5.5-6.5, equal volume of aluminum hydroxide is added to obtain aluminum adjuvant mixed liquid with the final concentration of 1mg/mL, the aluminum adjuvant mixed liquid is mixed evenly, the mixture is adsorbed for 1h at room temperature, and the mixture is stored at 4 ℃.
2. Preparation of rHBP V2 adsorption product with aluminum phosphate adjuvant
Taking the sterilized and filtered rHBP V2 protein prepared in example 2, adjusting the pH to 6.0-7.0, adding equal volume of aluminum hydroxide to obtain an aluminum adjuvant mixed liquid with the final concentration of 1mg/mL, mixing uniformly, adsorbing at room temperature for 1h, and storing at 4 ℃.
3. Preparation of rHBP V3 adsorption product with aluminum phosphate adjuvant
Taking the sterilized and filtered rHBP V3 protein prepared in example 2, adjusting the pH to 6.5-7.0, adding equal volume of aluminum hydroxide to obtain an aluminum adjuvant mixed liquid with the final concentration of 1mg/mL, mixing uniformly, adsorbing at room temperature for 1h, and storing at 4 ℃.
Diluting the prepared adsorption system of rHBP and aluminum phosphate adjuvant in 3 with 5mmol/L phosphate buffer solution (pH7.2), and then mixing 3 adsorption products in equal volume to obtain the final product with the following components: the contents of three variants of rHBP, V1, V2 and V3, were all 25.0. mu.g/ml, and the pH was adjusted to 7.2.
Example 5 Sterilization testing of group B epidemic meningococcal recombinant protein vaccine
Female BALB/c mice weighing 16-18g were randomly grouped into 10 mice each, and immunized with the group B recombinant meningococcal encephalitis protein vaccines prepared in examples 3 and 4, respectively, along with a saline control group. Injecting 0.5ml of rHBP V1, V2 and V3 into the abdominal cavity of each mouse, immunizing once on the 0 th day, the 21 st day and the 35 th day respectively, collecting blood in orbit on the 14 th day after the last immunization, separating serum, and carrying out related detection such as bactericidal activity by sterile subpackage.
1. TTC liquid preparation: preparing 0.5% (W/V) TTC solution with sterilized distilled water, heating to dissolve, placing in brown bottle, and storing at 2-8 deg.C for use.
2. Preparation of indicator bacterial suspension, group B epidemic meningococcus 341215 was inoculated on blood agar plates, 5% CO2Culturing at 37 deg.C for 16-18h, washing thallus Porphyrae with nutrient broth, measuring OD value at 600nm with spectrophotometer, adjusting OD value to 0.1 to obtain stock solution, and diluting at a ratio of 1:10000 (equivalent to 10)4cfu/mL) was used.
3. Complement-indicator suspension preparation: rabbit complement was diluted 1:20 with sterile PBS (pH7.4) or normal saline, and indicator bacteria were added to the final concentration of 1% (v/v).
4. Microtiter plates: the washing treatment of a 4X 10-well polystyrene plastic plate was performed by the same ELISA method. 1 drop (0.025mL) of PBS or physiological saline is respectively dropped into each hole of the titration plate by a micropipette in the vertical direction, and then a drop (0.025mL) of inactivated serum is added into the first hole of each row, and each plate can be used for preparing 4 specimens.
5. Complement addition-indicator suspension: diluting 2 times from the first hole of each row to the tenth hole, adding 1 drop of complement-indicator bacteria suspension into each hole, capping, shaking on a micro-oscillator for 90s, placing in a humid container, and culturing in a 37 deg.C water bath for 30 min.
6. Adding TTC nutrient broth: 0.15mL of nutrient broth containing 0.01% of TTC was added to each well, mixed well, and then placed in a humidified container and cultured in a 37 ℃ water bath for 4 hours.
7. And (4) observing results: the reaction plate is placed on white paper to observe results, and the titer of the group B epidemic meningococcal killing antibody of the specimen is detected by taking the hole with the highest serum dilution and no color change.
TABLE 1 TTC method for detecting group B epidemic meningococcal recombinant protein vaccine-elicited bactericidal antibodies
The improvement of the complement-mediated bactericidal antibody titer by 8 times or more has clinical significance, and the bactericidal power result of the group B epidemic meningococcal recombinant protein vaccine (table 1) shows that the antigens adsorbed by the two aluminum adjuvants can stimulate the generation of mouse anti-virus antibodies, wherein the bactericidal antibody titer generated by the group B epidemic meningococcal recombinant protein vaccine (aluminum phosphate adjuvant) is particularly obvious.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A group B epidemic meningococcus recombinant protein vaccine is characterized in that recombinant proteins containing three variants of human factor H binding protein are rHBP V1, rHBP V2 and rHBP V3 respectively;
wherein, the nucleotide sequences of the recombinant proteins for coding rHBP V1, rHBP V2 and rHBP V3 are respectively shown in SEQ ID NO. 1-3;
wherein,
the rHBP V1 recombinant protein is prepared by the following method: synthesizing rHBP V1 gene fragment, wherein the nucleotide sequence is shown as SEQ ID NO.1, adding Bgl II and Xho I enzyme cutting sites at the 5 'end and the 3' end of the gene respectively, connecting with an expression vector, introducing into host cells, and screening to obtain an engineering strain MH-rHBP V1 for efficiently expressing rHBP V1, wherein the preservation number is CGMCCNo.10718; fermenting and culturing the engineering strain, inducing to express recombinant protein, purifying, filtering and sterilizing;
the rHBP V2 recombinant protein is prepared by the following method: synthesizing rHBP V2 gene fragment, wherein the nucleotide sequence is shown as SEQ ID NO.2, adding Hind III and Xho I enzyme cutting sites at the 5 'end and the 3' end of the gene respectively, connecting with an expression vector, introducing into host cells, and screening to obtain an engineering strain MH-rHBP V2 for efficiently expressing rHBP V2, wherein the preservation number is CGMCC NO. 10719; fermenting and culturing the engineering strain, inducing to express recombinant protein, purifying, filtering and sterilizing;
the rHBP V3 recombinant protein is prepared by the following method: synthesizing rHBP V3 gene fragment, wherein the nucleotide sequence is shown as SEQ ID NO.1, Nde I enzyme cutting sites and Xho I enzyme cutting sites are respectively added at the 5 'end and the 3' end of the gene, the Nde enzyme cutting sites and the Xho enzyme cutting sites are connected with an expression vector, the Nde enzyme cutting sites and the Xho enzyme cutting sites are introduced into a host cell, and an engineering strain MH-rHBP V3 for efficiently expressing rHBP V3 is obtained by screening, wherein the preservation number is CGMCC NO. 10720; fermenting and culturing the engineering strain, inducing to express recombinant protein, purifying, filtering and sterilizing.
2. The vaccine of claim 1, wherein in the preparation of the rHBP V1, rHBP V2 and rHBP V3 recombinant proteins, the fermentation culture of 3 engineered strains expressing recombinant proteins is: respectively inoculating the engineering strains into LB liquid culture medium containing ampicillin with the final concentration of 50 mug/mL, culturing overnight at 37 ℃ to prepare seed liquid, transferring the seed liquid into liquid culture medium suitable for growth of escherichia coli to ferment: at 37 ℃, the pH value is 7.0-7.2 and the speed is 200 r/min.
3. The vaccine of claim 1, wherein the rHBP V1, rHBP V2, and rHBP V3 recombinant proteins are produced by inducing expression of the recombinant proteins by: when the bacteria grow to OD600 ═ 15, adding IPTG with final concentration of 0.1mmol/L for induction, controlling the induction temperature to be 30 ℃, continuing to culture for 6-8 h, stopping culture, continuously centrifuging at 8000rpm and 4 ℃ for 20min, collecting thalli, washing the thalli with Tris-HCl buffer solution with pH7.0 and 20mmol/L for 2 times, homogenizing and crushing, then centrifuging at 12000g and 4 ℃ for 30min, and discarding the precipitate.
4. The vaccine of claim 1, wherein the rHBP V1, rHBP V2, and rHBP V3 are present in the vaccine in an amount of 22.5 to 27.5 μ g/ml, and the pH is adjusted to 7.0 to 7.4.
5. The vaccine of claim 4, wherein the rHBP V1, rHBP V2 and rHBP V3 are each present in the vaccine in an amount of 25.0 μ g/ml, and the pH is adjusted to 7.2.
6. The vaccine of claim 1, further comprising an adjuvant selected from one or more of aluminum phosphate, aluminum hydroxide, and MF 59.
7. The vaccine of claim 6, wherein the aluminum content is 0.5-1.5mg/ml of vaccine.
8. A method of producing a group B epidemic meningococcal recombinant protein vaccine according to any one of claims 1 to 6, comprising the steps of:
(1) preparing rHBP V1, rHBP V2 and rHBP V3 recombinant proteins; the nucleotide sequences for coding the recombinant proteins are respectively shown as SEQ ID NO. 1-3;
the preparation method of rHBP V1, rHBP V2 and rHBP V3 recombinant proteins comprises the following steps: respectively connecting rHBP V1, rHBP V2 and rHBP V3 genes with a vector, transforming, selecting and identifying a correct positive clone, transferring the positive clone into a competent cell, selecting a monoclonal, inoculating a culture medium, and adding IPTG (isopropyl thiogalactoside) to induce and select a strain with the highest expression quantity to be used as an engineering strain for storage; the strains with the highest expression amount are respectively as follows: MH-rHBP V1 has a preservation number of CGMCC NO.10718, MH-rHBP V2 has a preservation number of CGMCC NO.10719, and MH-rHBP V3 has a preservation number of CGMCC NO. 10720;
(2) respectively fermenting and expanding the obtained 3 recombinant protein high-expression strains for culture, and inducing to express recombinant proteins; purifying protein, filtering and sterilizing;
(3) quantifying 3 recombinant proteins rHBP V1, rHBP V2 and rHBP V3, respectively adsorbing with adjuvant, ultracentrifuging to remove endotoxin, and mixing the three to obtain group B epidemic meningococcal recombinant protein vaccine.
9. The method of claim 8, wherein the adjuvant in step (3) is aluminum adjuvant, and the aluminum content is 0.5-1.5mg/ml after mixing the three.
10. The method of claim 8 or 9, wherein the rHBP V1, rHBP V2, and rHBP V3 are each present in the vaccine in an amount of 22.5 to 27.5 μ g/ml, and the pH is adjusted to 7.0 to 7.4.
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| CN102834410A (en) * | 2010-03-30 | 2012-12-19 | 奥克兰儿童医院及研究中心 | Factor h binding proteins (fhbp) with altered properties and methods of use thereof |
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| CN102028941A (en) * | 2011-02-11 | 2011-04-27 | 中国医学科学院医学生物学研究所 | Neisseria meningitidis group B recombinant protein chimeric vaccine and preparation method thereof |
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