阅读说明：本技术 一种B群流脑fHbp-V2重组蛋白及其制备方法，疫苗组合物，用途 (Group B epidemic encephalitis fHbp-V2 recombinant protein and preparation method thereof, vaccine composition and application ) 是由 刘建东 张静飞 徐颖之 张敬仁 刘建凯 郑海发 于 2019-12-20 设计创作，主要内容包括：本发明提供了一种B群流脑fHbp-V2重组蛋白及其制备方法,疫苗组合物,用途,所述疫苗抗原蛋白基因来自中国流行的B群流脑菌株,所述抗原蛋白免疫小鼠可以诱导产生平均滴度为102600的结合抗体反应和针对fHbp-V2和fHbp-V3的B群流脑菌株的杀菌抗体反应,可以用来预防fHbp-V2和fHbp-V3基因的B群流脑菌株导致的感染。(The invention provides a B-group epidemic encephalitis fHbp-V2 recombinant protein, a preparation method thereof, a vaccine composition and application thereof, wherein antigen protein genes of the vaccine are from Chinese epidemic B-group epidemic encephalitis strains, and an antigen protein immune mouse can induce and generate a binding antibody reaction with the average titer of 102600 and a bactericidal antibody reaction aiming at the B-group epidemic encephalitis strains of fHbp-V2 and fHbp-V3 and can be used for preventing infection caused by the B-group epidemic encephalitis strains of fHbp-V2 and fHbp-V3 genes.)

1. A group B epidemic cerebrospinal meningitis fHbp-V2 recombinant protein, which is characterized by having an amino acid sequence shown as seq ID No. 1.

2. A group B epidemic encephalitis fHbp-V2 gene sequence encoding the group B epidemic fHbp-V2 recombinant protein of claim 1.

3. The group B epidemic encephalitis fHbp-V2 gene sequence of claim 2, having the nucleotide sequence shown in seq id No. 2.

4. The group B epidemic encephalitis fHbp-V2 gene sequence of claim 2, wherein the nucleotide sequence synthesized after codon optimization according to the codon preference of Escherichia coli is shown as seq ID No. 3.

5. A vaccine composition comprising the group B epidemic encephalitis fHbp-V2 recombinant protein of claim 1 and, as adjuvant, aluminum hydroxide, complete freund's adjuvant, or incomplete freund's adjuvant.

6. The vaccine composition of claim 5, wherein said group B epidemic encephalitis fHbp-V2 recombinant protein is present at 200 ug/ml.

7. The vaccine composition of claim 6, wherein the aluminum hydroxide adjuvant is present in an amount of 1 mg/ml.

8. Use of the group B epidemic encephalitis fHbp-V2 recombinant protein antigen according to claim 1 in the preparation of a vaccine for preventing cerebrospinal meningitis or bacteremia diseases.

9. The method of claim 1, wherein the method of preparing a group B epidemic encephalitis fHbp-V2 recombinant protein comprises the steps of:

obtaining a gene sequence: the nucleotide sequence of fHbp-V2 gene shown in seq ID No.2 is obtained from the fHbp gene subtype V2.18 strain of the group B epidemic cerebrospinal meningitis preserved in the Chinese disease control center by a PCR gene amplification method, wherein PCR primers for amplification are respectively: an upstream primer: TGACCTGCCTCATTGATGC, downstream primer: GCCGTCCGAACACGATAATTTACCG, respectively;

codon optimization: carrying out codon preference optimization synthesis on the nucleotide sequence of the fHbp gene shown as seq ID NO.2 by using lasergene software to obtain the nucleotide sequence shown as seq ID NO. 3;

constructing a prokaryotic expression plasmid:

carrying out restriction enzyme digestion on the codon-optimized fHbp-V2 gene and a prokaryotic pET28a expression vector by using Nco I and EcoR I restriction enzymes, recovering gene fragments, then connecting the gene fragments by using T4 DNA ligase, converting a connecting product into a DH5 α competent cell, culturing and identifying;

constructing an expression strain:

successfully constructed clone transformed BL21 expression competent cells are induced, cultured and induced to express, and the expression of mycoprotein is identified by SDS-PAGE protein electrophoresis; screening strains with high expression quantity;

expression and purification of fHbp-V2 recombinant protein:

and culturing, collecting, crushing and purifying the strain with high expression level to obtain the fHbp-V2 recombinant protein.

Technical Field

The invention belongs to the field of vaccines, and particularly relates to a group B epidemic cerebrospinal meningitis fHbp-V2 recombinant protein, an amino acid sequence of the recombinant protein, a gene for encoding the recombinant protein, a vaccine composition containing the recombinant protein, a preparation method of the recombinant protein, and application of the recombinant protein in preparation of a vaccine for preventing cerebrospinal meningitis or bacteremia diseases.

Background

Epidemic cerebrospinal meningitis (epidemic cerebrospinal meningitis for short) belongs to the second infectious disease prescribed in China, is an infectious disease which is caused by gram-negative Neisseria and is transmitted through a respiratory tract with acute cerebrospinal meningitis and septicemia as main symptoms, infants and teenagers are main groups with diseases, Neisseria meningitidis usually does not cause diseases, and can be planted in the nasopharynx part of a human body in an asymptomatic mode, and cerebrospinal meningitis and bacteremia can be caused in some cases. The incidence rate of epidemic cerebrospinal meningitis in China is reduced to below 0.01/10 ten thousand, and the Chinese legal infectious disease data in 2018 issued by the Chinese disease control and prevention control center shows that the incidence rate of epidemic cerebrospinal meningitis in China is 0.0075/10 ten thousand, and the mortality rate is 8-15%.

According to the nature of its capsular polysaccharide, epidemic encephalitis can be divided into 13 serogroups, of which A, B, C, W and Y serogroups are the major epidemic groups, and more than 95% of cases of epidemic encephalitis are caused by infection with these serogroups. Among them A, C, W, Y group epidemic encephalitis corresponding capsular polysaccharide vaccine and polysaccharide conjugate vaccine are already on the market, and are used for preventing the infection of the four epidemic encephalitis serogroups. And the B-group epidemic encephalitis is low in immunogenicity because the capsular polysaccharide is an analogue of sialyl acyl protein of human cells, and cannot be used for researching the B-group epidemic encephalitis vaccine.

The fHbp is lipoprotein which is positioned on the outer membrane of the epidemic encephalitis and can be combined with the H factor, the H factor has an important function in a complement system, and the fHbp can escape from the killing effect of an immune system on the epidemic encephalitis strain in a mode of being combined with the H factor and is an important group B epidemic encephalitis vaccine antigen. Based on the protein sequence of fHbp, fHbp is divided into 3 variant groups V1, V2, and V3, with the homology of fHbp antigen within each variant group being 91.6-99%, and the homology of fHbp antigen between different variant groups being at least 62.8%. According to the research data of the Chinese disease control center on the B-group epidemic encephalitis, the fHbp genes of the B-group epidemic encephalitis strains in China and European and American areas are different, the fHbp gene of the European and American epidemic strains is mainly V1 and accounts for 70% of the epidemic strains, and the fHbp gene of the Chinese epidemic B-group epidemic encephalitis strain is mainly V2.

2B-group epidemic encephalitis vaccine products are on the market in European and American areas, one vaccine comprises two fHbp antigen proteins, namely fHbp-V1.45 and fHbp-V3.55, the other vaccine also comprises fHbp-V1.1 protein, and the three fHbp proteins are not found in the Chinese B-group epidemic encephalitis strain, so that the fHbp antigen protein for screening the Chinese epidemic B-group epidemic encephalitis strain plays an important role in developing the vaccine according with the Chinese B-group epidemic situation.

Disclosure of Invention

The invention provides a group B epidemic cerebrospinal meningitis fHbp-V2 recombinant protein which has an amino acid sequence shown as seq ID No. 1.

The invention provides a group B epidemic encephalitis fHbp-V2 gene sequence which encodes the group B epidemic encephalitis fHbp-V2 recombinant protein as claimed in claim 1.

Preferably, the nucleotide sequence of the group B epidemic cerebrospinal meningitis fHbp-V2 gene sequence is shown as seq ID No. 2.

Preferably, the nucleotide sequence of the group B epidemic cerebrospinal meningitis fHbp-V2 gene is synthesized after codon optimization according to the codon preference of Escherichia coli, and is shown as seq ID No. 3.

The invention provides a vaccine composition which is characterized by comprising the group B epidemic cerebrospinal meningitis fHbp-V2 recombinant protein and aluminum hydroxide serving as an adjuvant, a complete Freund adjuvant or an incomplete Freund adjuvant.

Preferably, the content of the recombinant protein of the group B epidemic cerebrospinal meningitis fHbp-V2 is 200 ug/ml.

Preferably, the content of the aluminum hydroxide adjuvant is 1 mg/ml.

The invention provides application of the group B epidemic cerebrospinal meningitis fHbp-V2 recombinant protein antigen in preparation of a vaccine for preventing cerebrospinal meningitis or bacteremia diseases.

The invention provides a preparation method of the group B epidemic cerebrospinal meningitis fHbp-V2 recombinant protein, which comprises the following steps:

obtaining a gene sequence: the nucleotide sequence of fHbp-V2 gene shown in seqID No.2 is obtained from fHbp gene sub-variant V2.18 strain of group B epidemic cerebrospinal meningitis preserved in Chinese disease control center by PCR gene amplification method, wherein PCR primers used for amplification are respectively: an upstream primer: TGACCTGCCTCATTGATGC, downstream primer: GCCGTCCGAACACGATAATTTACCG, respectively;

codon optimization: carrying out codon preference optimization synthesis on the nucleotide sequence of the fHbp gene shown as seq ID NO.2 by using lasergene software to obtain the nucleotide sequence shown as seq ID NO. 3;

constructing a prokaryotic expression plasmid:

carrying out double enzyme digestion on the codon-optimized fHbp-V2 gene and a prokaryotic pET28a expression vector by using Nco l and EcoR I restriction endonucleases, recovering gene fragments, then connecting the gene fragments by using T4 DNA ligase, converting a connecting product into a DH5a competent cell, culturing and identifying;

constructing an expression strain:

successfully constructed clone transformed BL21(DE3) expression competent cells are induced, cultured and induced to express, and SDS-PAGE protein electrophoresis is used for identifying the expression of mycoprotein; screening strains with high expression quantity;

expression and purification of fHbp-V2 recombinant protein:

and culturing, collecting, crushing and purifying the strain with high expression level to obtain the fHbp-V2 recombinant protein.

The fHbp-V2 recombinant protein antigen gene sequence related by the invention is derived from a strain of a B-group epidemic encephalitis in China, the fHbp gene of the strain belongs to a sub-Variant type V2.18(Variant 2.18 from the Chinese disease prevention and control center), the antigen gene is subjected to codon optimization synthesis according to the codon preference of escherichia coli and then cloned to a prokaryotic expression vector pET28a, the B-group epidemic encephalitis fHbp-V2 recombinant protein antigen is obtained after expression and purification, and immune serum of the obtained fHbp-V2 antigen can generate a bactericidal antibody aiming at the B-group epidemic encephalitis strain of fHbp-V2 and fHbp-V3, and the effect is obvious.

The fHbp-V2 recombinant protein antigen can induce cross bactericidal antibody reaction aiming at the group B epidemic encephalitis fHbp-V3 strain, and can be used for preventing infection of the group B epidemic encephalitis fHbp-V2 strain and the group B epidemic encephalitis fHbp-V3 strain.

The invention has the advantages that the antigen gene of the fHbp-V2 recombinant protein obtained by screening is from epidemic B-group epidemic encephalitis strain in China, and can be used for preventing the epidemic of the epidemic B-group epidemic encephalitis strain in China.

Drawings

FIG. 1: a DNA electrophoresis diagram of the fHbp-V2 gene sequence by double enzyme digestion;

FIG. 2: a DNA electrophoresis picture of pET28a carrier double enzyme digestion;

FIG. 3: an SDS-PAGE picture of the expression condition of the prokaryotic expression protein strain of the fHbp-V2 gene;

FIG. 4: an SDS-PAGE profile of the purified fHbp-V2 recombinant protein;

FIG. 5: the binding antibody response induced by the immune serum of the recombinant protein fHbp-V2;

FIG. 6: the FHbp-V2 recombinant protein immune serum induces a bactericidal antibody response;

Detailed Description

In order that those skilled in the art will better understand the technical solutions of the present invention, the present invention will be further described in detail with reference to the following detailed description.

Experimental methods without specific conditions noted in the examples of this patent generally follow conventional conditions, such as molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).