CN107080841B

CN107080841B - Cholera-rotavirus combined vaccine based on recombinant carrier protein and preparation method thereof

Info

Publication number: CN107080841B
Application number: CN201710258940.4A
Authority: CN
Inventors: 史晋; 马涛; 王文灏; 李津
Original assignee: Bravovax Co ltd
Current assignee: Bravovax Co ltd; SHANGHAI BOWO BIOTECHNOLOGY CO Ltd
Priority date: 2017-03-30
Filing date: 2017-04-19
Publication date: 2020-07-10
Anticipated expiration: 2037-04-19
Also published as: CN107050443A; CN107098976A; CN107050443B; CN107098976B; CN107080841A

Abstract

The invention discloses a recombinant carrier protein-based cholera-rotavirus combined vaccine and a preparation method thereof, wherein the combined vaccine comprises a.cholera vaccine comprising escherichia coli heat-labile enterotoxin B subunit (L TB) protein, vibrio cholerae enterotoxin B subunit protein (CTB) recombinant protein and cholera antigen, and b.rotavirus vaccine, wherein the rotavirus vaccine and the cholera vaccine are suspended for clinical use.

Description

Cholera-rotavirus combined vaccine based on recombinant carrier protein and preparation method thereof

Technical Field

The invention relates to a cholera-rotavirus combined vaccine based on recombinant carrier protein and a preparation method thereof, belonging to the field of biomedicine.

Background

Pathogenic microorganism and vaccine

Microorganisms that cause infectious diseases in the human or animal body are called pathogenic microorganisms or pathogenic microorganisms. Infection refers to a series of pathophysiological processes caused by the growth and reproduction of pathogenic microorganisms in certain parts after invading the body. After the pathogenic microorganism invades the organism, the pathogenic microorganism interacts with the organism to mutually change the activity and function of the other, so whether infectious diseases can be caused depends on the pathogenic capability, namely pathogenicity or virulence, of the pathogenic microorganism and the resistance, namely immunity, of the organism. The magnitude of the ability of a pathogenic bacterium to cause infection is the virulence or pathogenicity of the bacterium. The presence or absence of virulence and the strength of virulence of a bacterium depend mainly on its invasiveness, toxigenicity and ability to cause hypersensitivity reactions.

Toxins produced by bacteria can be divided into two broad categories, exotoxins and endotoxins. Exotoxins are metabolites that are secreted by pathogenic bacteria into the surrounding environment during growth and reproduction, and are produced primarily by gram-positive bacteria, and by a small number of gram-negative bacteria. The chemical composition of the protein is protein, has strong antigenicity and toxicity, but is extremely unstable, sensitive to heat and certain chemical substances and easy to damage. Common are as follows: diphtheria exotoxin produced by corynebacterium diphtheriae, tetanus toxin produced by clostridium tetani, enterotoxin produced by vibrio cholerae, botulinum toxin produced by clostridium botulinum, and the like. Most gram-negative bacteria produce endotoxin, which is actually present in the outer layer of the bacterial cell wall, which is a constituent of the cell wall, is not normally secreted into the environment, and is released only when the bacteria are lysed, and is therefore called endotoxin, which is less toxic and less antigenic than exotoxins.

Different individuals of the same organism may be ill when they come into contact with pathogenic bacteria, and may be safe and unharmed because of the differences in immunity among individuals. Immunity refers to a protective response in which the body recognizes and eliminates antigenic foreign bodies (e.g., pathogenic microorganisms). It is generally beneficial to the body, and can also damage the body under abnormal conditions. The immunity of the human body is classified into non-specific immunity and specific immunity. Wherein specific immunity refers to the specific resistance produced by the body against a certain microorganism or a certain class of microorganisms or products. The vaccine is a biological product developed by scientists to ensure that organisms generate specific immunity and resist the invasion of pathogenic microorganisms to human bodies, and is prepared by the pathogenic microorganisms. The pathogenic microorganisms such as bacteria, viruses, rickettsia and the like are prepared into vaccines, and after the vaccines are injected into organisms, the organisms generate specific or sensitized lymphocytes to secrete antibodies, so that the specific immune effect is achieved.

Vaccines are classified into therapeutic vaccines and prophylactic vaccines, in which diseases are treated by therapeutic vaccines and the body is protected from pathogenic microorganisms by prophylactic vaccines. Over the course of many years of effort, the medical community has developed a variety of vaccines to prevent various diseases caused by infections such as bacteria, viruses and fungi, which have greatly improved the health level of humans. The continuous development of biotechnology promotes the diversification of vaccine varieties. Vaccines developed by inactivated virus technology, such as Japanese encephalitis vaccine, poliomyelitis vaccine, influenza vaccine and the like, are used for preventing infectious diseases caused by viruses; attenuated live vaccines developed by attenuated virus technology, such as rotavirus vaccine, oral poliovirus vaccine, measles virus vaccine, mumps virus vaccine, rubella virus vaccine, varicella vaccine and the like. The bacterial vaccines developed by the purification technology of biological macromolecules such as useful proteins and polysaccharides for preventing bacterial infectious diseases, such as tetanus toxoid, diphtheria toxoid, pertussis toxoid, subcellular components thereof, epidemic meningococcal polysaccharide, 23-valent pneumococcal polysaccharide, and the like. More advanced bacterial vaccines that prevent meningitis and pneumonia, such as haemophilus epidemic type b polysaccharide-protein conjugate vaccines, pneumococcal polysaccharide-protein conjugate vaccines with a valence of 7 or 10, and meningococcal polysaccharide-protein conjugate vaccines with a valence of 4, are developed using semi-chemical conjugation techniques. Through the continuous improvement of biotechnology, more novel vaccine products can be developed to cope with the challenges of human health by different pathogenic microorganisms.

Second, mucosal immunization

Unlike the conventional immune system, the mucosal immune system is a separate immune system with unique structure and function, and has positive significance for preventing and treating pathogen colonization and invasion, wherein a large amount of immune cells and immune molecules are dispersed in the mucosal epithelium or the inherent layer under the mucosa (dispersed lymphoid tissue) or mucosa-associated lymphoid tissue formed by single or multiple lymphoid follicles, more than 50% of the lymphoid tissue and more than 80% of the immune cells are concentrated in the mucosal immune system, and mucosal immunity can induce the local mucosa to produce protective antibodies such as secretory IgA (sIgA), IgM and IgG, and can induce the mucosa of other parts to produce sIgA, which is the main mechanism of the mucosal immune protection, and further induces the immune system L to react with the IFN-4C and produce IFN-4C⁺T cells, which are important for the prevention and clearance of pathogen invasion. Mucosal immunity is therefore an important barrier to protect the body from pathogens and is of great importance in the design of vaccines.

Vaccines based on mucosal immunity tend to have weak response and short duration due to the induced immunity, and are difficult to achieve ideal immune protection effects. It is now believed that the lack of immunogenicity of antigens such as recombinant proteins, synthetic polypeptides and DNA is one of the important reasons, and therefore efforts are made to increase the intensity of the immune response, and that there are also some vaccines that require a shift in the type of immune response to highlight mucosal immunity and the like. These problems make the use of adjuvants urgent and important, so the research on mucosal immune adjuvants has become a hot research point in the fields of infection immunity and vaccines. At present, the reported mucosal immunoadjuvants can be mainly divided into three categories: the first is bacterial material, including proteins (primarily bacterial toxins) and nucleic acids; the second is various cytokines; the third category is antigen delivery systems.

Among the bacterial toxins, the most commonly used mucosal immunoadjuvant is E.coli heat-labile enterotoxin (L T) and Cholera Toxin (CT), which have been much studied, the closely linked toxins (Zout), cytotoxic necrosis factor (CNF 1), and dermonetic toxin (DNT), etc., are recently reported bacterial toxins with mucosal adjuvant function, L T and CT are both bacterial toxins with about 80% homology in nucleotide sequence and substantially identical structure, and CT mainly induces antigen-specific CD4⁺Th2 type cells, L T induced mixed CD4⁺Th1 and Th2 cell CT and L T are potent mucosal immunoadjuvants, but because of their toxicity, they have prevented their use in the field of human vaccines, and it is therefore essential to construct mutants that eliminate or reduce toxicity, while retaining adjuvant properties.

L T is one of the most effective mucosal immunogens that has been identified to date in human and animal experiments, it is effective in mediating CD4 against L T⁺L T is injected into mice by a digestive tract route (oral or intragastric route) to induce high secretory and systemic antibody responses, a large amount of IgA antibodies resisting L T can be detected in respiratory secretions and respiratory contents of the mice, respiratory mucosa is taken as a section, a large amount of plasma cells can be seen in the leucoderma of the mucosa lamina propria, the most intense mucosal secretory antibody response induced by L T is an antigen deposition site, but not limited to the antigen deposition site, and the same responses occur in other mucosal effector sites.

L T and L TB both have good immunogenicity, and L TB contains most of the dominant epitopes causing T-specific antibody responses, both can effectively start the body to generate local and systemic T cell and B cell immune responses, and can also enable T, B cells to generate long-term memory response.

Coli and epidemiology thereof

Escherichia coli is a short name for Escherichia coli (e.coli), isolated from infant feces by the german microbiologist, Escherichia, in 1885 at the earliest, and is a member of the enterobacteriaceae (enterobacteriaceae) and Escherichia (Escherichia), formerly known as bacillus coli commune, meaning a common Bacterium in the intestinal tract. In fact, only a few e.coli strains can directly cause host disease in the gut, while most e.coli are not pathogenic in the gut, but can cause extra-intestinal infection if translocated to tissues or organs outside the gut. Coli is not a pathogenic pathogen (e.g., reduced resistance, imbalance or shift in the proportion of normal flora, etc.), but rather, escherichia coli of some serotype groups (types) is itself a pathogenic bacterium. For intestinal pathogenic escherichia coli, there are mainly the following types depending on the pathogenic mechanism, clinical symptoms, epidemiological characteristics, and the like: enterotoxigenic e.coli (ETEC), Shiga toxin-producing e.coli (STEC), Enterohemorrhagic e.coli (EHEC), Enteropathogenic e.coli (EPEC), Enteroinvasive e.coli (EIEC), and the like. In addition, there are uropathogenic e.coli (UPEC), abscission e.coli (AEEC), necrotoxic enterobacter coli (NTEC), enteroadhesive e.coli (EAEC), and Enteroaggregative e.coli (EAggEC).

The most common of these is ETEC-induced diarrhea. The first epidemic of ETEC in the late 60's of the 20 th century is one of the important pathogens of infectious diarrhea in humans and animals. Some data indicate that ETEC is the most common cause of cholera syndrome in indigenous adult residents in the epidemic cholera; is the most common cause of diarrhea of children and travelers in developed countries and even developing countries; is also one of the important factors of diarrhea, nausea, low fever, abdominal cramp and the like caused by the pollution of water sources and food by excrement.

ETEC produces 2 different types of toxins, Heat-labile toxin (L T) and Heat-labile toxin (ST). L T and ST, which differ greatly in their physical properties, ST being essentially inactivated at 100 deg.C for 30min, but L T being completely inactivated at 65 deg.C for 30 min.ST has a low molecular weight (<9000ku) and no antigenicity and can be divided into two types, ST1 and ST2, where ST1 plays an important role in epidemiology, both of which activate guanylate cyclase, increase intracellular cGMP levels, disturb dielectric metabolism, and cause diarrhea. L T differs from ST in that L T is an immune protein closely related to cholera enterotoxin (Cholrea toxin, CT), constituting a toxin family.

Based on the source of toxin, L T can be divided into two human species of human (L T-h/h L0T) and pig (L1T-p/p L2T), which have very similar chemical properties and immunological properties, L3T is divided into two types, L4T 1 and L T2, L T1 is encoded by large plasmid in cytoplasm, most commonly L T is the main component for exerting biological activity, generally said L T is commonly referred to as L T1, and L T2 is encoded by chromosomal DNA, although their composition and mode of action are similar, they have no gene homology and cross immunogenicity, hereinafter said L T is referred to as L T1.

The gene encoding L T has a full length of 1148bp, is located on a large plasmid in the cell packaging of wild-type toxigenic Escherichia coli. L T operon contains 2 structural genes, respectively a toxB gene encoding a B subunit and a toxA gene encoding an A subunit, which are connected in series and transcribed into 1 mRNA chain, wherein the toxB gene is located at the 3' end of the toxA gene, its ribosome binding site (GGGA) is located in the toxA gene, and 2 codons at the toxA3' end (TTATGA) and 2 codons at the toxB 5' end (ATGAAT) have 4 nucleotides overlapping each other.A subunit coding region has a full length of 372bp, encodes a polypeptide consisting of 124 amino acids.A subunit coding region has a full length 777bp, encodes a polypeptide of 259 amino acids.toxB has a SD sequence 3 nucleotides before the start codon, wherein 5 nucleotides can complement 5 nucleotides of 16S ribosomal 3' end 12 nucleotides, and the ATG is complementary to a more specific amino acid sequence than the start codon, thus facilitating the expression of the toxin in the 16S ribosomal 3' S12 nucleotides, which it is more easily regulated by the toxA promoter, and the toxB promoter, which it is more easily than the toxA promoter, which it is located in the most easily.

A, B subunits, which are assembled into complete L T.B subunits after crossing cell membrane, are specifically bound to GM-1 ganglioside receptor on the surface of eukaryotic cell membrane to facilitate the A subunits entering target cells, the A subunits have GTP-dependent ADP-ribosylation transferase activity, which disturbs the degradation and equilibrium of cAMP in cells through G protein-mediated ADP-ribosylation reaction, stimulates the increase of cAMP content, thereby inducing toxic effects, L T-h and L T-p act on different G proteins, the former is Gs protein and the latter is Gi protein, and the final effect is to lead to the inactivation of protein kinase A, to cause a series of biochemical reactions, to increase cAMP content, and to stimulate the excessive secretion of intestinal mucosal water and electrolytes, thereby causing diarrhea.

L TB has been proven as the function of immunoadjuvant, and has higher and higher attention, but L TB has little research as carrier protein, and L TB protein alone is only used as L TA carrier at present, which greatly limits the application range of L TB L TB has a good structural basis, and protein carriers constructed after fusion expression with other proteins have a wide application prospect.

Vibrio cholerae and epidemiology thereof

Cholera is a severe intestinal infectious disease threatening the survival of human beings, and is one of the class A infectious diseases in China. Since its history, there have been seven global pandemics of cholera, each of which has led to hundreds of thousands or even millions of infections, with thousands of deaths. Today cholera is abusive in many countries worldwide.

Cholera is a severe intestinal infectious disease caused by vibrio cholerae and is characterized by severe painless diarrhea and vomiting, rice water sample stool, severe dehydration, muscle cramp, peripheral circulatory failure and the like in clinic. In the 60 s of the 20 th century, various parenteral cholera vaccines have been developed internationally, but because of severe adverse reactions, the protection is only within 50%, the protection period is short, and the WHO biological standardization specialist committee is determined to stop using the vaccine from 1999.

Oral vaccines induce antibodies in the gut, which is the front line for combating cholera. Furthermore, oral cholera vaccines have been developed in recent years because they reduce the cost of medical treatment and avoid unnecessary medical injuries.

The main oral cholera vaccines currently available are: inactivated whole cell cholera vaccine (WC vaccine), inactivated whole cell plus B subunit cholera vaccine (WC/rBs vaccine), and live attenuated cholera vaccine (CVD103-HgR live attenuated vaccine).

Inactivated whole cell vaccine (WC) was originally directed against O1 group cholera, and achieved a good protective effect in field trials in Vietnam (Tracch DD, Clemens JD, Ke NT, et al, field title of locally produced, killed, oral cholera vaccine in Viet Nam. L ancet, 1997, 349 (9047): 231. su 235.), with an effective rate of 66% after 8 months in each age group, but only in a few countries such as Vietnam and Indonesia, and subsequently, according to the recommendations of the WHO diarrhea research council, a2 nd generation inactivated whole cell bivalent vaccine was developed, consisting of O1 group and O139 whole cells, containing 5 × 1010cfu formalin 139 in which Vibrio cholera was studied orally in Hanoi at 1.5 m/dose, L, but with an undesirable immune effect at 2 weeks.

Cholera toxin B subunit-inactivated cholera vibrio whole-body vaccine (BS-WC) is added with cholera toxin B Subunit (BS) on the basis of WC vaccine. BS is a good immunogen superior to cholera toxoid but inferior to cholera toxin. A combined vaccine (BS-WC) consisting of BS and inactivated Vibrio cholerae whole-body vaccine (WC) is tested on site in the epidemic area of Bengal cholera in 1985-1989. 63498 the immune protection rate of people in the first 6 months after vaccination is 85%, and after 3 years, the immune protection rate is reduced to 51%, which proves that the BS-WC vaccine has good protection effect on cholera. The oral vaccine consisting of recombinant cholera toxin B subunit and inactivated O1 group cholera whole cells has achieved good results in preventing O1 group cholera, and thus the world health organization recommends for the vaccine. In the last 90 s the vaccine was approved for registered production in sweden. However, this vaccine requires the use of antacids for oral administration to neutralize gastric acid and therefore has a greater adverse effect. Moreover, in the outbreak of cholera, the method is lack of clean water source, and a large amount of water-soluble antacid is needed, thereby increasing the cost of the medicine virtually.

On the basis that the BS-WC cholera vaccine has good safety, immunogenicity and high protection rate as proved by human body experiments, the development work of the oral cholera vaccine is further advanced, namely recombinant BS (rBS) is adopted to replace natural cholera BS, and the rBS-WC cholera oral vaccine is constructed. Researchers such as horse Qingjun, etc. of bioengineering research institute of Chinese military medical science institute developed a new national medicine rBS-WC oral cholera vaccine. The oral cholera vaccine is an oral cholera vaccine independently developed by Chinese scientists and is one of the oral cholera vaccines formally recommended by the world health organization.

CT is a thermolabile enterotoxin secreted by Vibrio cholerae and consists of a toxic A subunit (CTA) and five identical B subunits (CTB) forming an AB5 structure. The A subunit is a single chain, which is often cleaved after synthesis between residues 194 and 195 to give CTA1 and CTA2, which are disulfide-linked. Wherein, the CTA1 has ADP-ribosyltransferase activity and is closely related to the adjuvant effect of CT, and the CTA2 is connected with the CTA1 and CTB. The 5 subunits of CTB are non-covalently bound to each other as pentamers, which in turn surround CTA via CTA2 to form AB5 structure. The binding force between subunits of CTB pentamer is strong, and is larger than that of a subunit.

The CTB is taken as a nontoxic unit, has the function of combining GM1, is that a toxic subunit A is tightly combined on the cell surface of intestinal mucosa, so that the fusion protein is easier to act with the digestive tract mucosa, a series of physiological and biochemical reactions are further caused, a stronger immune effect is generated, and the CTB can also be taken as a stable carrier of some exogenous polypeptides, and can arouse the immunogenicity of fusion epitope antigens after the CTB and the antigens which are chemically coupled or genetically fused enter the body simultaneously, so that the body generates strong immune reaction, and the immune protection effect is achieved.

CTB has strong immunogenicity, can enhance the immunogenicity of bacterial viruses and other antigens, particularly can enhance the specific IgG antibody response of serum and induce stronger specific mucosal IgA immune response through a mucosal immune path, and has the characteristic of enhancing the antigenicity of an epitope. CTBs can stimulate antigen trafficking ability of dendritic cells used to transport antigen to lymphoid organs in antigen presenting cells and activate antigenic CD4+ and CD8+ T cells. CTB can activate maturation of dendritic cells, making the dendrites of dendritic cells longer and more bulky. CTB cells may also regulate T cell responses within the line, as well as antibody production in vivo.

Penta, rotavirus and epidemiology thereof

Rotavirus is one of the major pathogens causing severe diarrhea in infants. About 1.11 million infants less than 5 years of age suffer from RV diarrhea worldwide each year, and about 35 to 59 million infants die of RV diarrhea each year, 82% of which occur in developing countries. Epidemiologically, there was no significant difference in the incidence of rotavirus in developed and developing countries, indicating that improving the sanitary environment of life had no significant effect on controlling rotavirus diarrhea. Currently, no specific medicine exists for diarrhea caused by RV, and vaccination becomes an effective and only means for preventing and controlling RV diarrhea. Three RV vaccines widely used at the present stage are attenuated live vaccines, although the morbidity and mortality of rotavirus infection can be effectively reduced, the potential risk of causing diseases still exists, and the vaccines are possible to recover virulence in a human body due to the occurrence of reverse mutation.

Rotavirus belongs to reoviridae, rotavirus. The diameter under an electron microscope is about 65-75 nm, the double-stranded RNA genome is non-enveloped and has a double-layer shell and 11 segments. The length of the gene is different from 680 to 3300bp, and the 11 genes encode 6 structural proteins (VP1, VP2, VP3, VP4, VP6 and VP7) and 6 non-structural proteins (NSP1 to NSP6) in total. VP4 and VP7 have type-specific epitopes that can produce specific neutralizing antibodies that induce protective immune responses, VP6 has group-specific epitopes, and NSP4 protein has enterotoxin, which is an important factor in diarrhea.

Human rotaviruses are very diverse. To date, the combination of at least 42 different P-G serotypes has been identified due to the free combination and isolation of G and P proteins, resulting in the production of different strains. Fortunately, only a few specific rotavirus strains have the ability to infect humans with global spread. International universal RV genotyping is determined by homology comparison of protein coding frame (ORF) nucleotide sequences. The commonly used sero (genotypic) typing of rotaviruses is mainly determined by the two structural proteins VP4 and VP 7. Rotaviruses are classified into 19 types G (serotypes) according to the VP7 gene; rotaviruses were classified into 28 types of P (genotypes) according to the VP4 gene. VP4 serotype, two types are currently found: g1, G3 and G4 are the same type and are P1A; g2 is P1B.

Rotarix (G1P type), Rotateq (G1-G4P type) and Rotewei (G10P type) from the Lanzhou institute of biologicals of our country are among the currently available rotavaccines in the market. Among them the rotavirus vaccines Rotarix and Rotateq were found in large clinical phase iii assessment studies of more than sixty thousand people in many countries without any risk of causing unwanted intussusception. However, the selling price of the two vaccines is slightly higher, and a large area of vaccination cannot be undertaken in many developing countries. And the Rotewei is only produced and sold in China, and the limitation is also foreseeable. The medical and health organizations of all countries are actively developing novel rotavirus vaccines, but no well-recognized breakthrough progress exists at present.

Because rotavirus and cholera virus both cause diarrhea reaction and have higher similarity of pathogenic environment, in order to reduce the economic burden and the inoculation frequency of vaccinees, a combined vaccine capable of jointly immunizing rotavirus and cholera virus is urgently needed to be developed.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a cholera-rotavirus combined vaccine based on recombinant carrier protein and a preparation method thereof.

In order to realize the aim, the technical scheme of the cholera-rotavirus combined vaccine based on the recombinant carrier protein and the preparation method thereof is as follows:

the invention aims at providing a cholera-rotavirus combined vaccine based on recombinant carrier protein, which comprises the following components in part by weight:

a. cholera vaccine comprises cholera antigen and antigen carrier, wherein the cholera antigen is coupled with the antigen carrier, the antigen carrier is recombinant protein carrier, and recombinant carrier protein is obtained by gene recombination of Escherichia coli heat-labile enterotoxin B subunit (L TB) protein and Vibrio cholerae enterotoxin B subunit protein (CTB);

b. a rotavirus vaccine;

rotavirus vaccine and cholera vaccine were suspended for immediate use.

L the TB nucleic acid sequence and the CTB nucleic acid sequence are bridged through a primer sequence, the constructed L TB-CTB nucleic acid sequence is shown as the sequence table SEQ ID NO. 5, and the coded carrier protein sequence is shown as the sequence table SEQ ID NO. 6.

L the nucleotide sequence of TB is connected with the nucleotide sequence of CTB by designing a primer, wherein the primer at end F of L TB nucleotide sequence is shown as SEQ ID NO. 7 of the sequence table, the primer at end R is shown as SEQ ID NO. 8 of the sequence table, the primer at end F of CTB nucleotide sequence is shown as SEQ ID NO. 9 of the sequence table, and the primer at end R is shown as SEQ ID NO. 10 of the sequence table.

Preferably, the CTB nucleic acid sequence is selected from the CDS region of the O139 group cholera complete gene sequence.

The nucleotide sequence of the encoded L TB protein is shown in a sequence table SEQ ID NO. 1, and the encoded amino acid sequence is shown in a sequence table SEQ ID NO. 2.

The nucleotide sequence of the CTB protein is shown in a sequence table SEQ ID NO: 3, and the coded amino acid sequence is shown in a sequence table SEQ ID NO: 4, respectively.

Preferably, the cholera antigen is a protein antigen and/or a polysaccharide antigen.

More preferably, the cholera antigen is a cholera capsular polysaccharide.

In a preferred embodiment, the cholera antigen is a cholera capsular polysaccharide of the group O139 of Vibrio cholerae.

More preferably, the mass ratio of the vibrio cholerae polysaccharide to the carrier protein is 0.4-0.8: 1 to carry out coupling.

The cholera vaccine is in any one of spray, liquid, capsule, lyophilized powder, tablet and pill.

The cholera-rotavirus combined vaccine based on the recombinant carrier protein and the preparation method thereof also comprise sucrose which is used as a freeze-drying protective agent of a freeze-dried preparation of the cholera vaccine.

Any component that can be used as a vaccine lyoprotectant is considered to fall within the scope of the present invention, which is not limited to sucrose.

Preferably, the rotavirus vaccine comprises a rotavirus inactivated vaccine or a rotavirus inactivated vaccine.

The second aim of the invention is to provide a cholera-rotavirus combined vaccine based on recombinant carrier protein and a preparation method thereof, which comprises the following steps:

s1, designing two pairs of primers according to CDS region nucleic acid sequences of L TB and CTB, constructing pET28 a-L TB-CTB plasmids, carrying out double enzyme digestion through BamH I and Xho I after PCR amplification, and recovering L TB-CTB fragments and an expression vector pET28a through gel;

s2, connecting and transforming an escherichia coli DH5 α strain, screening positive clones, performing monoclonal amplification, and identifying after IPTG induction;

s3, coupling the cholera antigen with the recombinant carrier protein, and detecting a binding product after coupling.

When vibrio cholerae polysaccharide is selected as the cholera antigen, S3 is specifically as follows:

coupling cholera polysaccharide and recombinant carrier protein, wherein the mass ratio of the polysaccharide to the carrier protein is (0.5-4): and 1, detecting the binding product after coupling.

When cholera protein is selected as the cholera antigen, S3 specifically is:

coupling cholera protein and recombinant carrier protein, wherein the mass ratio of the cholera protein to the monomer protein is 0.4-1: and 1, detecting the binding product after coupling.

Compared with the prior art, the combined vaccine provided by the invention has the advantages that the recombinant L TB-CTB protein carrier is coupled with the capsular polysaccharide of cholera O139 strain, and is temporarily suspended with a rotavirus vaccine, so that the serum immunity of an inoculated person can be caused, and the mucosal immunity in intestinal tracts can be caused.

Drawings

FIG. 1 is a schematic diagram of the construction of plasmid pET28 a-L TB-CTB provided by the present invention.

FIG. 2 is a schematic diagram of the plasmid for constructing pET28 a-L TB-CTB plasmid provided by the present invention.

FIG. 3 is a schematic diagram of the induction and purification results of recombinant L TB-CTB carrier protein provided by the invention.

Detailed Description

The present invention will be described more fully hereinafter with reference to the following examples. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were all commercially available unless otherwise specified.

Cloning and prokaryotic expression of recombinant L TB and CTB proteins

L T like CT et al, L T is a h mutexameric protein of the AB5 type consisting of A, B two subunits (L T-A and L T-B). A, B two subunits are non-covalently bound, and the individual subunits are not biologically active and only when bound together have the biological and chemical properties of the holotoxin.A subunit is the virulence center of the toxin, has a molecular weight of about 28KD, has ADP-ribosylating enzymatic activity.A subunit can be cleaved into two fragments, A1 (L T-A1) and A2 (L T-A2), A1 and A2 fragments, in a folded and helical structure, which are linked by a disulfide bond.A 1 is the active portion of L T toxin, A2 is the portion linked to the B subunit.when the disulfide bond between A1 and A2 is reduced, the enzymatically active A1 subunit is released.B subunit consists of two helical residues and the six residues of the helical subunit is linked together, and the N-cysteine residue is linked to the central cysteine residue of the subunit of the loop of GM 72. α, the cDNA residue linked to the end of the subunit, α A α, the loop of the subunit linked to form a α receptor molecule.

According to L TB and CTB nucleic acid sequences and amino acid sequences registered by GeneBank, the specific sequences are as follows:

l TB nucleic acid sequence (375bp) is shown in sequence table SEQ ID NO: 1, and the amino acid sequence (124a.a, MW 14133.74) coded by the nucleic acid sequence is shown in sequence table SEQ ID NO: 2.

The CTB nucleic acid sequence (375bp) is shown as a sequence table SEQ ID NO: 3, the CTB amino acid sequence (124 a.a.mw. 13896.46) encoded by the nucleic acid sequence is as shown in SEQ ID NO: 4, respectively.

pET28a was selected as a vector, and primers were designed based on the full-length CDS sequences of L TB and CTB registered in GeneBank as follows:

l TB-Fwd comprises a protection base-BamH I enzyme cutting site-L TB protein N-terminal sequence from 5 'end to 3' end, and the primers (shown as SEQ ID NO: 7 in the sequence table) are as follows:

5’-CG--GGATCC--atgaataaagtaaaatgttatgttttatttacggcgtta-3’

l TB-Rev comprises a protective base, an Eco311 enzyme cutting site, a CTB protein N-terminal sequence, a L TB protein C-terminal sequence from a 5 'end to a3' end, and primers (shown as a sequence table SEQ ID NO: 8) are as follows:

5’-CTAG--GGTCTC--cat--gtttttcatactgattgccgcaa-3

the CTB-Fwd sequentially comprises a protective base, an Eco311 enzyme cutting site, a L TB protein C-terminal sequence, and a CTB protein N-terminal sequence from a 5 'end to a3' end, wherein the primers (shown as a sequence table SEQ ID NO: 9) are as follows:

5’-CTAG--GGTCTC--AAC--atgattaaattaaaatttggtgttttttttacagtttta-3’

the CTB-Rev comprises the following components from 5 'end to 3' end: protecting base- -Xho I cleavage site- -CTB protein C terminal sequence. The primers (shown as SEQ ID NO: 10 in the sequence table) are as follows:

5’-CC--CTCGAG--ttaatttgccatactaattgcggcaa-3’

as shown in figures 1-2, pET28 a-L TB-CTB plasmid is constructed, because L TB and CTB have higher homology, a fragment of CTB is inserted into a L TB fragment, the fragment is connected by primers, and PCR is carried out by bridging, so that a full-length fragment of the recombinant L TB-CTB carrier protein can be obtained by amplification, the complete sequence of the constructed pET28a plasmid is shown as a sequence table SEQ ID NO. 5, and the coded amino acid sequence is shown as a sequence table SEQ ID NO. 6.

After the vector and the PCR product are subjected to double enzyme digestion by BamHI and XhoI, L TB and CTB fragments are recovered from gel, after the gel is recovered, DNA ligase is used for connecting overnight at 16 ℃, Escherichia coli DH5 α is connected and transformed, monoclonal amplification is carried out, after plasmids are extracted, about 800bp bands identified by double enzyme digestion of BamHI and XhoI are screened under ampicillin resistance, sequencing is carried out after positive screening, and sequencing results are compared on NCBI websites, so that the results are completely correct.

Secondly, induced expression and purification of recombinant L TB-CTB carrier protein

The recombinant L TB-CTB carrier protein is induced for 30h at 20 ℃, the concentration of IPTG is 0.3 mmol/L, the induction condition is optimal, after induction, a proper amount of supernatant and precipitate of an ultrasonic cracking product are removed for SDS-PAGE, meanwhile, protein before induction is used as a contrast, after bacteria breaking, the expression protein is found to exist in the form of an inclusion body, after the inclusion body is washed, the inclusion body is dissolved by 8 mol/L of urea, after renaturation of renaturation liquid, the recombinant fusion protein with the purity of about 93 percent is obtained by purification through a glutathione S-transferase (GST) affinity chromatographic column, the SDS-PAGE result shows that an additional band with about 30kD appears in the recombinant bacteria cracking precipitate and is similar to the expected result, because the protein is mainly expressed in the form of the inclusion body, the long-time induction at a lower temperature is selected, the purification result of the inducible expression is shown in a graph 3, a band 1 is a protein, 2 is a total Marker before induction, 3 is a total protein after induction, 4 is a supernatant after bacteria breaking, 5 is a bacterial fragment, and 5 is a centrifugal carrier protein after centrifugation, L is a recombinant TB-CTB carrier protein after induction.

Results experiment of recombinant L TB-CTB carrier protein linked with cholera lipopolysaccharide (L PS)

3.1 extraction of cholera lipopolysaccharide

The lipopolysaccharide molecule consists of an O-side chain, a core oligosaccharide and a lipid A, wherein the O-side chain is a long chain with a repeating structure of oligosaccharide consisting of 2-4 glycosyl groups, the core oligosaccharide is a branched oligosaccharide chain consisting of 9-10 glycosyl groups, the lipid A part is a framework of lipopolysaccharide, and the basic composition unit is D-glucosamine disaccharide connected with β -1, 6-glycosidic bonds, and the lipopolysaccharide on the cell wall of vibrio cholerae is a thallus antigen (O antigen) which is a virulence factor and an important protective antigen.

In this example, O139 serotype V.cholerae was selected to extract lipopolysaccharide, the bacterial cells were expanded and cultured, 95% phenol was added, ultracentrifugation was performed to collect the supernatant, absolute ethanol was added to the supernatant until the ethanol concentration was 70%, centrifugation was performed overnight at 4 ℃ to discard the supernatant, and the transparent gelatinous precipitate was dissolved in 2 mmol/L MgSO₄Adding DNase and RNase into 50 mmol/L Tris (pH7.6) buffer solution, placing the solution in a warm box overnight, adding 15mg proteinase K, transferring the solution into a dialysis bag, dialyzing at room temperature for 24h, wherein the dialysate is the buffer solution, placing the dialysis bag into water after enzyme hydrolysis, dialyzing at 4 ℃ for 2d, changing water every 12h, centrifuging the dialyzed solution, dissolving the precipitate in a small amount of water, and freeze-drying for storage to obtain purified L PS.

The molecular weight of the purified L PS is confirmed to be correct by detection, the total nucleic acid content in the L PS sample is less than or equal to 5 percent, and the residual protein content is less than or equal to 2 percent.

3.2 conjugation of cholera lipopolysaccharide to recombinant L TB-CTB Carrier protein

The freeze-dried powder of the recombinant L TB-CTB carrier protein is dissolved to the required concentration after being added with water for injection and is reserved after being dissolved.

The lyophilized lipopolysaccharide was diluted with water for injection, 60 μ L CDAP (100mg/M L in acetonitrile) was added to 1M LL PS (10mg/M L), pH was controlled at 5.8-6, reaction was carried out for 30s, 140 μ L aqueous TEA solution was added to pH7.0, reaction was carried out for 2min, 1M L5 mg/M L solution of recombinant L TB-CTB carrier protein was added, pH was adjusted to 8.0 with 0.1M NaOH, rotation was carried out overnight at 4 ℃, and 100 μ L ethanolamine solution was added to terminate the reaction.

3.3 detection of binding products

Purifying the combined product by a gel filtration chromatography column, wherein the eluent is 0.2M NaCl, detecting the light absorption value at the ultraviolet 280nm position, and collecting substances in each elution peak.

And (3) confirming that the combined product contains recombinant L TB-CTB components by adopting an SDS-PAGE method, and detecting the protein content, and detecting the polysaccharide content by adopting a phenol-sulfuric acid method to confirm that the polysaccharide content in the product is about 95 mu g/m L.

Detecting whether the binding product is combined with GM1 on the surface of the intestinal tract or not through GM1-E L ISA to stimulate a mucous membrane system to generate mucous membrane IgA.

1) Coating, diluting GM1 to 10 μ g/m L with carbonate coating buffer, adding 100 μ L of the above coating antigen to each reaction well of a 96-well plate, standing overnight at 4 deg.C, removing the solution in the well, and washing 3 times with washing buffer PBST;

2) blocking, adding 4% BSA solution 200 μ L into the coated reaction well, standing at 37 deg.C for 2 hr, discarding blocking solution, and washing for 3 times;

3) adding CTB solution and conjugate sample solution with a series of dilution gradients, wherein each well is 100 mu L, incubating at 37 ℃ for 1.5 hours, and washing for 3 times;

4) adding primary antibody, namely adding 100 mu L of rabbit anti-CTB IgG antibody diluted by 1: 4000 into each hole, and incubating for 2 hours at 37 ℃;

5) adding enzyme-labeled antibody, namely adding 100 mu L of freshly diluted enzyme-labeled donkey anti-rabbit IgG antibody into each reaction well, incubating for 1 hour at 37 ℃, and washing for 3 times;

6) adding substrate solution for color development, namely adding 100 mu L of TMB substrate solution into each reaction hole, and standing for 10-15 minutes at room temperature;

7) stopping the reaction, namely adding 2M sulfuric acid 50 mu L into each reaction hole;

8) reading: and detecting the light absorption value of each hole at the ultraviolet 450nm position on a microplate reader, and printing and recording.

The OD value of the recombinant L TB-CTB protein fluctuates between 0.25 and 0.48, which indicates that the recombinant L TB-CTB in the binding product still maintains the binding capacity with the GM1, and the capacity is stable, so that good mucosal immune response can be caused.

3.4 in vivo assay

Carrying out living body infection on the successfully-connected binding product, carrying out intraperitoneal injection administration on the successfully-connected binding product, selecting healthy BA L B/c mice for 6-8 weeks for testing, wherein each group contains 10 mice, the administration amount of each group is 1mg, the first group is administered with the binding product, the second group is administered with unloaded recombinant L TB-CTB carrier protein, the third group is administered with CT protein, the fourth group is administered with L T protein, the fifth group is administered with physiological saline as negative blank control, the administration is carried out for 3d at 30 ℃, the systemic reaction of the mice is continuously observed within 3-7 d, the first sampling is carried out after 7d, each group is killed by 2 mice for sampling every 7 days, serum and intestinal mucus are collected during sampling, food is cut off for 24h before each sampling, water is not cut off, so as to reduce intestinal contents, the serum is taken to detect the serum IgG antibody level of each group of mice, and each group is killed by 1 mouse to detect the IgA antibody level in the intestinal mucus.

Preparation of serum: collecting orbital blood of a mouse, standing the collected blood at 37 ℃ for 2h, centrifuging the blood at 3000r/min for l0min, sucking serum, storing the blood at low temperature and preparing for detection.

Mice sacrificed after blood collection, immediately dissected and approximately 10cm of jejunal midsection isolated. Weighing empty tube in advance, scraping intestinal mucus, placing in the tube, weighing the total weight, subtracting the empty tube weight from the total weight to obtain the scraped mucus weight, diluting with 4 times of PBS, storing at low temperature, and testing.

The indirect E L ISA is adopted to detect the IgG content in blood and the IgA content in intestinal tract, the detection result of the IgG content in blood is shown in the following table 1, and the detection result of the IgA content in intestinal tract is shown in the following table 2:

TABLE 1 measurement results of IgG content in blood

	7d	14d	21d	28d	35d
						First group	0.413	0.608	1.003	1.011	0.907
Second group	0.132	0.159	0.274	0.351	0.237
						Third group	0.375	0.559	0.868	0.694	0.501
Fourth group	0.486	0.604	0.815	0.733	0.472
						Fifth group	0.015	0.006	0.027	0.014	0.013

TABLE 2 IgA content test results in intestinal tract

	7d	14d	21d	28d	35d
						First group	0.231	0.328	0.698	0.814	0.788
Second group	0.112	0.149	0.195	0.188	0.191
						Third group	0.203	0.298	0.385	0.317	0.337
Fourth group	0.215	0.307	0.362	0.345	0.271
						Fifth group	0.006	0.011	0.030	0.051	0.004

Note: each group of data is obtained by repeated experiments, and the data results of each parallel group have no significant difference through statistical analysis.

As shown in tables 1 and 2, the recombinant L TB-CTB carrier protein has no toxicity of vibrio cholerae or escherichia coli, cannot directionally activate specific immune reaction, and compared with the simple CT protein and the L T protein, the recombinant antigen protein connected with lipopolysaccharide has stronger antibody titer, short initiation time of immune reaction, good immune effect and long duration of immune reaction.

3.5 quantitative production of binding products

And (3) combining the purified lipopolysaccharide and the recombinant L TB-CTB carrier protein in a mass ratio of 0.5-4: 1 in the step 3.2 to obtain a combined product, purifying and checking the combined product, adding sucrose as a freeze-drying protective agent, wherein the initial mass percentage of the sucrose in the freeze-drying stock solution is not more than 20%, and freeze-drying to obtain the recombinant cholera vaccine freeze-drying preparation.

The freeze-drying step specifically comprises the following steps:

1. preparing sucrose mother liquor with concentration of 70% and sterilized at 121 ℃ for 15 min;

2. placing the separated and purified pneumococcal vaccine obtained in the embodiment 1 into a sterile container, adding the sucrose mother liquor prepared in the step 1 to enable the concentration of sucrose to reach 10%, uniformly mixing to prepare a virus stock solution semi-finished product containing a sucrose protective agent, and filling the virus stock solution semi-finished product into penicillin bottles according to the specification of 1.0m L/bottle respectively to carry out subsequent freeze-drying process;

3. pre-freezing the semi-finished product obtained in the step 2, rapidly cooling to-55 ℃ during pre-freezing, and maintaining for 15-20 hours;

4. and (4) drying the semi-finished product obtained after pre-freezing in the step (4) in the first stage: heating to-45 to-40 ℃ for 50h, and heating to-40 to-33 ℃ for 22 h;

5. and (4) drying the semi-finished product obtained after the first-stage drying in the step (4) in the second stage: setting the temperature to be 0-30 ℃ in different time, and continuously maintaining for 28h in different temperature raising stages; and then preparing the recombinant cholera vaccine with the sucrose concentration of 10% in the freeze-dried stock solution, wherein the preparation method of the freeze-dried vaccine can refer to the prior art and is not described herein again.

Immunological evaluation of Combined rotavirus-cholera vaccine

4.1 preparation of Rotavirus cholera combination vaccine

Because the combined vaccine of the invention improves the combined immunity of rotavirus and vibrio cholerae, and improves the resistance of an vaccinee to enterovirus by causing strong mucosal immunity, the embodiment can adopt the prior art to prepare the rotavirus vaccine, and the rotavirus vaccine comprises inactivated rotavirus vaccine and inactivated rotavirus vaccine.

In this example, Vero cell culture vaccine rotavirus P2G 3 strain is selected, and after virus propagation, purification is carried out to obtain P2G 3 virus particles with complete infectivity, and virus genome is not changed by detection, and residual Vero cell DNA content is not more than 500 pg.

The purified P2G 3 virus liquid is used as the re-dissolved liquid of cholera conjugated product freeze-dried protein, that is, the P2G 3 virus liquid is added into the freeze-dried preparation of cholera vaccine conjugated product based on recombinant carrier protein before use, the conjugated product is re-dissolved and mixed evenly for intramuscular injection.

4.2 immunological testing of Rotavirus cholera combination vaccine

4.2.1 antigen detection of Rotavirus

The selected rotavirus strain is the group A rotavirus, and the kit is used for detecting the wild group A rotavirus antigen. Enzyme linked immunosorbent assay (EIA) is used for detecting whether a stool sample contains rotavirus antigen. The specimen is first thawed, then sampled and dissolved by adding virus preservation solution, and finally manipulated in the reaction well according to the EIA kit instructions.

The mice are divided into three groups for test, each group has 3 parallels, each parallels has 10 mice, the test result takes the average value of each group parallels, the first group is inoculated with the rotavirus cholera combined vaccine, the second group is inoculated with the rotavirus P2G 3 strain, the third group has negative control, the excrement of the mice is collected within 14 days after the vaccine inoculation, each group takes about 5-10G or about 5-10 m L, EIA test is carried out in the kit, the number of the mice with diarrhea of each group is counted, the average diarrhea rate is calculated, and the test result is shown in Table 3.

TABLE 3 EIA test results

As shown in Table 3, the rotavirus cholera combination vaccine of this example elicits a stronger immune response than the commercial rotavirus vaccine, and the antagonism or competition relationship between the two vaccines is very small. Meanwhile, strong discomfort of vaccinees (except for immunodeficiency patients) can not be caused by inoculation, and the diarrhea rate of the combined vaccine is similar to that of a commercial vaccine, but the time for generating adverse reactions is short, namely the detoxification period is short.

4.2.2 antibody titer detection of combination vaccines

The detection step is the same as the 3.4 in vivo test step, and the two tests are different only in the type of administration. The cholera vaccine was selected exclusively for enteric-coated capsules (Oravacs) when compared to dosing. Is administered orally.

The indirect E L ISA is adopted to detect the IgG content in blood and the IgA content in intestinal tract, the IgG antibody titer detection result in blood is shown in the following table 4, and the IgA antibody titer detection result in intestinal tract is shown in the following table 5:

TABLE 4 IgG antibody titer in blood test results

TABLE 5 IgA content test results in intestinal tract

As can be seen from the detection results, the conventional vaccine sold in the market has similar level with the combined vaccine in the invention in the aspect of serum antibody titer, but rarely can cause mucosal immunity, and the specific antibody generated in short time is few, so that the rotavirus-cholera live vaccine in the invention causes enough immune response in the mucosal layer, and stimulates the organism to generate enough specific antibody with long response time. Has better immune effect.

4.2.3 stability testing of combination vaccines

The combined vaccine is stored for 4 weeks at 4 ℃ for 12 months or 37 ℃, no abnormity is found in appearance inspection and identification tests, the pH value and the antigen content are kept relatively stable, and no obvious difference in antibody level is found in a mouse test. The cholera-rotavirus combination vaccine using the recombinant carrier protein is proved to have good stability when stored at 4 ℃ for 12 months or 37 ℃ for 4 weeks.

Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.

Claims

1. A recombinant carrier protein-based cholera-rotavirus combination vaccine, comprising:

a. cholera vaccine comprises cholera antigen and antigen carrier, wherein the cholera antigen is coupled with the antigen carrier, the antigen carrier is recombinant protein carrier, the recombinant carrier protein is obtained by gene recombination of escherichia coli heat-labile enterotoxin B subunit (L TB) protein and vibrio cholerae enterotoxin B subunit protein (CTB), the sequence of the carrier protein is shown in sequence table SEQ ID NO. 6, and the cholera antigen is polysaccharide antigen;

b. a rotavirus vaccine;

rotavirus vaccine and cholera vaccine were suspended for immediate use.

2. The cholera-rotavirus combined vaccine based on recombinant carrier protein of claim 1, wherein the L TB-CTB nucleic acid sequence is shown in SEQ ID NO. 5 of the sequence table, and the L TB nucleic acid sequence is bridged with the CTB nucleic acid sequence through a primer sequence.

3. The recombinant carrier protein-based cholera-rotavirus combination vaccine of claim 1, wherein: the CTB nucleic acid sequence is selected from the CDS region of the O139 group cholera complete gene sequence.

4. The cholera-rotavirus combined vaccine based on recombinant carrier protein as claimed in claim 1, wherein the nucleotide sequence of the encoded L TB protein is shown as SEQ ID NO. 1 of the sequence table, and the encoded amino acid sequence thereof is shown as SEQ ID NO. 2 of the sequence table.

5. The recombinant carrier protein-based cholera-rotavirus combination vaccine of claim 4, wherein: the nucleotide sequence of the CTB protein is shown in a sequence table SEQ ID NO: 3, and the coded amino acid sequence is shown in a sequence table SEQID NO: 4, respectively.

6. The recombinant carrier protein-based cholera-rotavirus combination vaccine of claim 1, wherein: the cholera antigen is cholera capsular polysaccharide of vibrio cholerae O139 group.

7. The recombinant carrier protein-based cholera-rotavirus combination vaccine of claim 1, wherein: the rotavirus vaccine comprises rotavirus inactivated vaccine or rotavirus attenuated vaccine.

8. The cholera-rotavirus combination vaccine based on recombinant carrier protein and the preparation method thereof according to claim 1, wherein the steps comprise:

9. The cholera-rotavirus combination vaccine based on recombinant carrier protein and the preparation method thereof of claim 8, wherein S3 is specifically: