阅读说明：本技术 一种包含B群脑膜炎球菌fHBP抗原的组合物及其制备方法与应用 (Composition containing group B meningococcus fHBP antigen and preparation method and application thereof ) 是由 胡浩 陈瑞勤 蒋浩然 于旭博 于 2019-11-08 设计创作，主要内容包括：本发明涉及生物医学领域,具体而言,涉及一种包含B群脑膜炎球菌fHBP抗原的组合物及其制备方法与应用。所述组合物包含B群脑膜炎球菌fHBP的变体1、变体2、变体3的重组蛋白V1、V2和V3；其中V1、V2和V3的氨基酸序列依次如SEQ ID NO:1～3所示,且V1、V2和V3均具有脂质修饰。该组合物相对于现有疫苗具有更广的抗原谱,能够覆盖更多类型的变体菌株,免疫效果好。(The invention relates to the field of biomedicine, and particularly relates to a composition containing a group B meningococcus fHBP antigen, and a preparation method and application thereof. The compositions comprise recombinant proteins V1, V2, and V3 of variant 1, variant 2, variant 3 of a group B meningococcal fHBP; the amino acid sequences of V1, V2 and V3 are sequentially shown as SEQ ID NO 1-3, and V1, V2 and V3 are all modified by lipid. Compared with the existing vaccine, the composition has a wider antigen spectrum, can cover more types of variant strains, and has a good immune effect.)

1. A composition comprising a group B meningococcal fHBP antigen, characterised in that it comprises recombinant proteins V1, V2 and V3 of variant 1, variant 2, variant 3 of a group B meningococcal fHBP;

the amino acid sequences of V1, V2 and V3 are sequentially shown as SEQ ID NO 1-3, and V1, V2 and V3 are all modified by lipid.

2. The composition comprising a group B meningococcal fHBP antigen of claim 1, wherein the molar ratio of V1, V2 and V3 in the composition is (1-3): (0.5-1.5): (0.5 to 1.5).

3. The composition comprising a group B meningococcal fHBP antigen of claim 2, wherein the molar ratio of V1, V2 and V3 in the composition is 2: 1: 1.

4. the composition of claim 2, comprising a group B meningococcal fHBP antigen, wherein the composition further comprises an adjuvant.

5. The composition of claim 4, comprising a group B meningococcal fHBP antigen, wherein the adjuvant is at least one of an aluminium salt adjuvant, a liposome, MF59, monophosphoryl lipid A, flagellin, CpG-ODN, and Poly (I: C).

6. A process for the preparation of a composition comprising a group B meningococcal fHBP antigen according to any one of claims 1 to 5, comprising the steps of:

obtaining recombinant plasmids which respectively comprise coding genes capable of expressing V1, V2 and V3;

respectively transforming the recombinant plasmids into host cells, identifying, screening and identifying correct positive strains, and transforming the correct positive strains to expression cells; culturing;

v1, V2 and V3 were isolated and enriched from the expression cells and mixed with optional adjuvants.

7. The method of claim 6, wherein the genes encoding V1, V2 and V3 are expressed as:

a) sequentially shown as SEQ ID NO. 4-6; or

b) A sequence which encodes the same protein as the nucleotide sequence of a), but differs from the nucleotide sequence of a) due to the degeneracy of the codons.

8. The method of claim 6 or 7, wherein the enrichment is affinity chromatography.

9. The method of claim 6 or 7, wherein the host cell and/or expression cell is a prokaryotic cell.

10. An antibody against meningococcus group B, prepared by immunizing an animal with a composition comprising a meningococcal group B fHBP antigen of any one of claims 1 to 5.

Technical Field

The invention relates to the field of biomedicine, and particularly relates to a composition containing a group B meningococcus fHBP antigen, and a preparation method and application thereof.

Background

Neisseria meningitidis (Neisseria meningitidis) is a gram-negative capsular bacterium that colonizes the upper respiratory tract in about 10% of the population. Based on the structure of the capsular polysaccharide, meningococci can be divided into 13 serogroups. Of these, A, B, C, Y and W135 are the major pathogens, causing over 95% of cases. Meningitis caused by group B strains in China, Europe and America and other countries exceeds 1/3, and becomes a main pathogenic bacterium. A, C, W135 and group Y, capsular polysaccharide can be used as vaccine antigen for preparing vaccine for preventing diseases. However, this method cannot be used for serogroup B. Because the capsular polysaccharide polysialic acid polymer is a human autoantigen, the capsular polysaccharide can cause cross reaction.

With the development of technologies such as genomics, proteomics, reverse vaccinology and the like, a series of vaccine candidate proteins which are positioned on the surface of bacteria and can induce bactericidal antibodies are screened and identified. Factor F binding protein (fHBP) is one of the most major protective antigens. This lipoprotein is expressed in all meningococcal serogroups and has been found to be present in a number of meningococcal strains. fHBP sequences can be divided into three families (variants V1, V2 and V3). Antisera raised against variant V1 was found to be able to confer bactericidal activity against strains within the subgroup V1, but not against variants V2 and V3. For variants V2 and V3, the reverse is true. Based on this, the united states Pfizer company developed a bivalent MenB vaccine (Trumenba) for the prevention of group B meningococci based on two variants of fHBP (V1 and V3). However, foreign vaccines are primarily directed against circulating strains in their native range. In China, the variant V3 also accounts for a certain proportion. Although mutant V2 has high homology with V3 and exhibits a certain cross-reactivity, the cross-protection effect is not satisfactory. In order to provide a broader protective effect, variant V3 may be necessary to incorporate a vaccine component to cope with the diversity and variant of group B meningococcal fHBP, thereby providing a more comprehensive protective effect for group B meningococcus. In addition, the fHBP antigen provided by the prior art has the problem of insufficient immunogenicity.

Disclosure of Invention

Based on the above, the invention aims to provide a composition containing group B meningococcal fHBP antigen and a preparation method thereof, wherein the composition has simple preparation method, can stimulate the body to produce antibodies aiming at different variant strains, and has stronger immunogenicity.

In order to achieve the purpose, the specific technical scheme of the invention is as follows:

a composition comprising a group B meningococcal fHBP antigen comprising recombinant proteins V1, V2 and V3 of variant 1, variant 2, variant 3 of a group B meningococcal fHBP;

the amino acid sequences of V1, V2 and V3 are sequentially shown as SEQ ID NO 1-3, and V1, V2 and V3 are all modified by lipid.

The invention also provides a process for the preparation of a composition comprising a group B meningococcal fHBP antigen as described above, comprising the steps of:

obtaining recombinant plasmids which respectively comprise coding genes capable of expressing V1, V2 and V3;

respectively transforming the recombinant plasmids into host cells, identifying, screening and identifying correct positive strains, and transforming the correct positive strains to expression cells; culturing;

v1, V2 and V3 were isolated and enriched from the expression cells and mixed with optional adjuvants.

The invention also provides an antibody for resisting the meningococcus group B, and the specific technical scheme is as follows:

an antibody against group B meningococcus, prepared by immunising an animal with a composition comprising a group B meningococcal fHBP antigen, as described above.

Compared with the prior art, the invention has the beneficial effects that:

the composition provided by the invention has three antigens of fHBP variants, and the antigen parts all have P4 signal peptide which can guide and locate the fHBP sequence on the cell membrane of the bacteria in the process of translation expression in bacteria, and then the signal peptide can be degraded and removed by a protease carried in the bacteria, and the fHBP mature peptide located on the cell membrane can be subjected to lipid modification. The lipid part can be used as an adjuvant component for enhancing the immunogenicity of fHBP, so that the immune effect is improved, and the use effect of the lipid part as a vaccine is better; and the selected fragment is modified to obtain soluble protein, which is more beneficial to expression and purification. And compared with the existing vaccine, the composition has a wider antigen spectrum and can cover more types of variant strains.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is an SDS-PAGE identification of three fHBPs in one embodiment of the invention; lane 1 is protein marker; arrows in lanes 2-4 indicate the purified fHBP recombinant proteins to variants 1, 2, 3, respectively;

FIG. 2 is a graph showing the WB assay results of three fHPBs reacted with corresponding sera, respectively, in one example of the present invention; lanes 1-3 arrows indicate the results of reaction of the fHBP recombinant proteins of variants 1, 2 and 3, respectively, with the corresponding variant sera;

FIG. 3 shows the detection of mouse serum antibody titer using purified recombinant protein (fHBP-V1/V2/V3) in one embodiment of the present invention; the histograms reflect the individual immune group antigen-specific serum antibody levels detected by the different coated variant fHBP antigens, respectively.

Detailed Description

In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It will be appreciated that the experimental procedures for the following examples, where specific conditions are not indicated, are generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The various reagents used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The present invention relates to a composition comprising a group B meningococcal fHBP antigen, comprising recombinant proteins V1, V2 and V3 of variant 1, variant 2, variant 3 of a group B meningococcal fHBP;

the amino acid sequences of V1, V2 and V3 are sequentially shown as SEQ ID NO 1-3, and V1, V2 and V3 are all modified by lipid.

In some embodiments, the molar ratio of V1, V2, and V3 in the composition is (1-3): (0.5-1.5): (0.5 to 1.5).

In some embodiments, the molar ratio of V1, V2, and V3 in the composition is (1.5 to 2.5): (0.7-1.3): (0.7 to 1.3).

In some embodiments, the molar ratio of V1, V2, and V3 in the composition is 2: 1: 1.

in some embodiments, an adjuvant is also included in the composition.

In some embodiments, the adjuvant is at least one of an aluminum salt adjuvant, a liposome, MF59, monophosphoryl lipid A, flagellin, CpG-ODN, and Poly (I: C). Preferably, the adjuvant is an aluminium hydroxide adjuvant or an aluminium phosphate adjuvant, or a combination of an aluminium hydroxide adjuvant or an aluminium phosphate adjuvant with other adjuvants suitable for use in humans. Among them, although freund's adjuvant cannot be used in human vaccines, it is understood that it can be used as an adjuvant for group B meningococcal vaccines for mouse trials in research.

According to one aspect of the invention, the invention also relates to a process for the preparation of a composition comprising a group B meningococcal fHBP antigen as described above, comprising the steps of:

obtaining recombinant plasmids which respectively comprise coding genes capable of expressing V1, V2 and V3;

respectively transforming the recombinant plasmids into host cells, identifying, screening and identifying correct positive strains, and transforming the correct positive strains to expression cells; culturing;

v1, V2 and V3 were isolated and enriched from the expression cells and mixed with optional adjuvants.

In some embodiments, the genes encoding V1, V2, and V3 are capable of expressing:

a) sequentially shown as SEQ ID NO. 4-6; or

b) A sequence which encodes the same protein as the nucleotide sequence of a), but differs from the nucleotide sequence of a) due to the degeneracy of the codons.

In some embodiments, the method of enrichment is affinity chromatography.

According to one aspect of the invention, the invention also relates to an antibody against group B meningococcus, which is prepared by immunizing an animal with a composition comprising a group B meningococcal fHBP antigen, as described above.

The subject to be immunized may be selected from animals including humans and all animal breeds (e.g., domestic animals and pets) and wild and avian birds including, without limitation, cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, turkeys, banisters, and the like.

The terms related to the present invention are explained as follows:

adjuvant: is a substance which is injected into an animal body simultaneously with or in advance of an antigen and can nonspecifically enhance the immune response of the body to the antigen, or is called a nonspecific immunopotentiator.

Aluminum salt adjuvant: the adjuvant prepared by the traditional process by taking aluminum salt as a material mainly comprises 3 aluminum phosphate, aluminum hydroxide and aluminum potassium sulfate, and the currently commonly used aluminum salt adjuvants are aluminum hydroxide and aluminum phosphate.

Liposome adjuvant: artificial cell membrane-like globules composed of lipid, single-layer or multi-layer bimolecular water-holding structure mainly composed of phospholipoid, cholesterol, stearylamine, etc. Can include various vaccines, and can be effectively introduced into cells, so that the stay time in the body is prolonged, the dosage of the vaccines is reduced, the toxic and side effects are reduced, and the immune function is improved.

TLR adjuvants: novel vaccine adjuvants developed based on ligands of Toll-like receptors, such as bacterial Lipopolysaccharide (LPS) as a ligand of TLR4, flagellin as a ligand of TLR5, unmethylated CpG sequences of bacteria or viruses as a ligand of TLR9, and other TLR ligands with adjuvant activity, are also included.

MF 59: an oil-in-water emulsion comprises squalene, sorbitol trioleate (Span85), tween 80 and citric acid buffer.

MPL: monophosphoryl lipid a, a TLR4 ligand, is one of the successfully developed TLR-like adjuvants.

CpG-ODN: artificially synthesizing oligodeoxynucleotide sequence containing unmethylated cytosine and guanine dinucleotide, which is a TLR9 ligand.

Poly (i.c): is a synthetic double-stranded RNA molecular analogue and is a TLR3 ligand.

SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Native-PAGE: native polyacrylamide gel electrophoresis.

ELISA: enzyme-linked immunosorbent assay.

SBA: and (5) detecting the bactericidal activity.

Embodiments of the present invention will be described in detail with reference to examples.