CN107961370B

CN107961370B - Multivalent pneumococcal conjugate vaccine and preparation method thereof

Info

Publication number: CN107961370B
Application number: CN201710183984.5A
Authority: CN
Inventors: 艾智武; 袁军; 程超; 张凡; 吴克
Original assignee: Bravovax Co ltd
Current assignee: Bravovax Co ltd; SHANGHAI BOWO BIOTECHNOLOGY CO Ltd
Priority date: 2017-03-22
Filing date: 2017-03-24
Publication date: 2020-08-11
Anticipated expiration: 2037-03-24
Also published as: CN107961368A; CN107961369B; CN107151270B; CN107151270A; CN107961370A; CN107961368B; CN107961369A

Abstract

The invention provides a multivalent pneumococcal conjugate vaccine based on a recombinant delta fHbp-NadA fusion protein carrier and a preparation method thereof. The two active proteins are fused and expressed by designing a soft connecting peptide chain, and then the two active proteins are made into a single protein while keeping respective activity, so that the method has more controllability in industrial production and immunization, greatly reduces the risk, and can provide more polysaccharide-protein binding sites.

Description

Multivalent pneumococcal conjugate vaccine and preparation method thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a multivalent pneumococcal conjugate vaccine and a preparation method thereof.

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.

Polysaccharide-protein conjugate vaccine and protein carrier thereof

Polysaccharides are an important immunologically active ingredient in pathogenic bacteria, and are classified into the fungal polysaccharides (OPS) and Capsular Polysaccharides (CPS). When pathogens invade the body, they act as immunogens which stimulate the body to produce a protective immune response. Many of the pathogens that are currently responsible for serious diseases, such as Haemophilus influenzae type B (Hib), Streptococcus pneumoniae (Spn) and Neisseria meningitidis (Nm), have polysaccharides on their surface that confer resistance to complement-mediated killing and phagocytosis, thereby promoting their survival in the blood during pathogenesis. The first generation of vaccines against Hib, Spn and Nm use polysaccharides as antigens. Unfortunately, these polysaccharide vaccines are not immunogenic in young children and do not produce immunological memory. This is because the polysaccharide molecule belongs to a T cell independent antigen (Ti-Ag), and is less immunogenic, and the immune effect is particularly undesirable after vaccination of infants. To improve the immunogenicity of polysaccharide vaccines, in the 20 th century and in the 30 th century, were enhanced by landstein, Avery and Goebel by coupling to proteins. In 1980, John Robbins and rachel schnerson described conjugates of Hib polysaccharide with diphtheria and tetanus toxoid proteins, greatly enhancing antibody responses in animal models. Finally the Hib polysaccharide-protein conjugate elicits a memory-type antibody response in human infants. The new generation of polysaccharide-protein conjugate vaccines has created a resurgence in vaccinology. Such conjugates transform past T cell-independent polysaccharide vaccines into T cell-dependent vaccines with greater immunogenicity in children, have been shown to have the ability to produce antibodies with high avidity, establish immunological memory, and produce a group immune effect. In addition, they improve the protective response of the immature immune system in young infants and the aging immune system in the elderly, thereby achieving optimal immune effects.

To date, 5 carrier proteins have been used in licensed conjugate vaccines: diphtheria toxin non-toxic variant (CRM197), Tetanus Toxoid (TT), meningococcal outer membrane protein complex (OMP), Diphtheria Toxoid (DT) and haemophilus influenzae protein d (hid). Clinical trials have demonstrated the efficacy of these conjugate vaccines in the prevention of infectious diseases and in drugs that alter Hib, Spn and Nm. All 5 carrier proteins are effective in increasing the immunogenicity of vaccines, but they give rise to different amounts and affinities of antibodies, the ability to carry multiple polysaccharides in the same product, and the ability to act simultaneously with other vaccines.

CRM197 is a non-toxic variant of the diphtheria toxin isolated from a culture of corynebacterium diphtheriae C7(β 197). CRM197 differs from the wild-type diphtheria toxin in that the point mutation at amino acid position 52 replaces glycine with glutamic acid, which eliminates its enzymatic activity and toxicity. CRM197 is antigenically indistinguishable from diphtheria toxin, but has the advantage of being a conjugate protein: non-toxic, and have more lysyl side chains available for conjugation. Another form of CRM used as a conjugate is a purified native diphtheria toxin, which is subsequently detoxified with formaldehyde, known as Diphtheria Toxoid (DT), should not be confused with CRM 197. TT is prepared from tetanus toxin produced by Clostridium tetani culture by formaldehyde detoxification. OMP is Nm serotype B outer membrane protein complex. DT is diphtheria toxin produced by the culture of corynebacterium diphtheriae, and is prepared by formaldehyde detoxification. HiD is the surface protein of Haemophilus influenzae purified and separated by SDS-PAGE step in the Haemophilus influenzae which is treated by ultrasonic wave initially, but the current vaccine obtains recombinant protein by genetic engineering.

TT, DT and other toxoids have been produced industrially, are easily available, and can greatly enhance the immunogenicity of polysaccharide after being coupled with polysaccharide. However, to obtain toxoids, bacterial toxins need to be chemically detoxified. In the process, point-to-point mutation or unnecessary modification can be generated, so that the physicochemical property of the toxin is changed, important T cell epitope is damaged, and the immunogenicity of the T cell epitope is influenced. This problem is avoided if a non-toxic variant toxin CRM197 is used. However, toxoid antibodies (TT and DT) present in the body are used in large amounts and may cause problems such as hypersensitivity or inhibition of the anti-carbohydrate reaction (immunosuppression). These potential problems are avoided by using polysaccharides and carrier proteins from bacteria of the same species. For example, the development of Nm vaccines, group B protein or OMP protein-based vectors for the preparation of A, C, Y, W135 group polysaccharide-protein conjugate vaccines are a good strategy, which in theory prevents almost all pathogenic Nm, provided that the group B protein vector is "broad-spectrum" or has significant cross-reactivity. Therefore, the development of novel protein carriers capable of conferring true protective immunogenicity on vaccines is at hand.

Third, the research on the hazard of pneumococcus and the epidemiology thereof

Infections caused by pneumococcus are a major cause of pneumonia morbidity and mortality worldwide, and have become an important public health problem worldwide. Pneumococci have become the first killer of children worldwide. The fatality rate of pneumonia in China is 16.4%, wherein the fatality rate of middle-aged and old people over 50 years old and infants under 1 year old is respectively as high as 28.6% and 22.0%. According to the difference of immunogenicity of capsular polysaccharide, Spn can be divided into 93 serotypes, but most serotypes rarely cause diseases, at least 70% -75% of invasive pneumonia worldwide is caused by 13 common serotypes, and national statistics show that the first serotypes of pneumococcal infection strains are sequentially: 5.

type

6, 19, 23, 14, 2, 4, Spn vaccines are important means to prevent their infection. 23-valent pneumococcal vaccines produced by China biotechnology group Chengdu biological product research institute are prepared by selecting 23 most common pathogenic bacteria (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F), fermenting, culturing, separating and purifying various polysaccharides on pneumococcal capsules respectively, and mixing the polysaccharides in equal proportion to prepare the vaccine. Clinical use proves that the vaccine prepared from the capsular polysaccharide is definitely effective and widely used in a plurality of countries. However, the polysaccharide vaccine has the problems of low affinity of generated antibodies, only 50-70% of protection rate on invasive pneumococcus, only generation of transient immunity, no immunological memory, no protection effect on infants under 2 years old and the like. The main reasons are that pure polysaccharide antigen can not activate T cells and induce immunological memory effect, and antibodies generated by the body are mainly IgM and IgG2 and can not effectively activate the complement system.

Thus, polysaccharide-protein conjugate vaccines against pneumococcus have been produced. Through technical development for many years, in 2000, 7-valent pneumococcal polysaccharide-CRM 197 conjugate vaccine successfully developed and marketed by the American Huishh (Wyeth) company is a multivalent vaccine prepared by respectively covalently bonding 7 different pneumococcal serotype polysaccharides to CRM197 protein carriers and mixing the polysaccharides to prevent infantile pneumonia, and the 7 serotype pneumococci cover more than 90% of the different serotype strains of pneumococci prevalent in North America and Europe. In the same year, the first 7(4, 6B, 9V, 14, 18C, 19F and 23F) valent pneumococcal protein conjugate vaccines of the feverfew company in the united states are on the market, and currently, the feverfew company is registering 13 valent conjugate vaccines in china, adding 6 serotypes (1, 3, 5, 7F, 6A, 19A). However, epidemiology in China shows that the serotypes of pneumococcal infection strains are sequentially as follows: 5. the coverage rate of 6, 1, 19, 23, 14, 2 and 4 types of pneumococcal conjugate vaccine with the price of feverfew 7 on common pathogenic bacteria in China is only about 50 percent, and the coverage rate of 13-price conjugate vaccine is only 70 percent. The conjugate vaccine developed abroad is not suitable for preventing the pneumonia of children in a large range in China.

There is also a non-negligible problem that as the number of different serotypes increases and the number of carrier proteins of the same species increases, the immunogenicity decreases. Clinical tests show that when the multivalent pneumococcal conjugate vaccine is inoculated with other univalent vaccines or multiple vaccines containing the same components with protein carriers, such as Hib-TT, diphtheria-Hib polysaccharide conjugate vaccine-IPV (inactivated polio vaccine) -hepatitis B vaccine (6-combination vaccine for short), the immunogenicity of most of serotype polysaccharides in the multivalent pneumococcal conjugate vaccine can be inhibited, and particularly, the immunogenicity of the serotype polysaccharides using tetanus toxoid as carriers is obviously influenced. The reason is that the total concentration of tetanus toxoid and diphtheria toxoid used as carriers in the multivalent pneumococcal conjugate vaccine is too high, and when the combined vaccine containing the diphtheria toxoid and the tetanus component are simultaneously inoculated, a carrier receptor competitive inhibition effect is caused, and the immunogenicity of a polysaccharide part of the conjugate vaccine is reduced. With the large use of the five carrier proteins (CRM197, TT, OMP, DT and HiD) currently on the market, the development of new carrier proteins is imminent. On the basis, 23-valent polysaccharide-protein conjugate vaccine suitable for Chinese epidemiology is developed, has great significance and social value, and protects infants in China from pneumonia caused by Spn infection.

Disclosure of Invention

In view of the above, the present invention provides a recombinant Δ fHbp-NadA fusion protein, which has stronger MenB flora immunogenicity and can induce strong serum bactericidal response in vivo. Meanwhile, the delta fHbp-NadA is used as a protein carrier, and a strong immunogenic conjugate vaccine capable of covering A, B, C, Y and W135 floras is provided.

The invention provides a recombinant delta fHbp-NadA fusion protein carrier, which comprises a recombinant delta fHbp, a flexible connecting peptide segment and NadA, wherein the recombinant delta fHbp comprises V of fHbp variable V1_A、V_BDomains and V of fHbp variant V3_C、V_D、V_EA domain.

The amino acid sequence of the recombinant delta fHbp is shown in a sequence table SEQ ID NO: 1, and the nucleotide coding sequence is shown in a sequence table SEQ ID NO: 2, respectively.

The nucleotide coding sequence of the flexible connecting peptide segment is shown in a sequence table SEQ ID NO: 3, respectively.

The amino acid sequence of the recombinant delta fHbp-NadA fusion protein carrier is shown in a sequence table SEQ ID NO: 4, the nucleotide coding sequence is shown in a sequence table SEQ ID NO: 5, respectively.

The second aspect of the present invention provides a preparation method of the recombinant Δ fHbp-NadA fusion protein vector, comprising the steps of:

s1, designing primers to respectively amplify to obtain a fHbp V1 gene fragment and a fHbp V3 gene fragment, performing bypass PCR amplification by taking the two gene fragments as templates to obtain a recombinant delta fHbp full-length fragment, recovering the delta fHbp gene fragment and a pET vector, and preparing a recombinant plasmid pET-delta fHbp;

s2, designing a full-length CDS sequence of the NadA protein through primer amplification, recovering a NadA gene fragment and a recombinant plasmid pET-delta fHbp, and preparing the recombinant plasmid pET-delta fHbp-NadA;

s3, converting the recombinant plasmid pET-delta fHbp-NadA into an expression strain to obtain the recombinant delta fHbp-NadA fusion protein vector.

The third aspect of the invention provides an application of the recombinant delta fHbp-NadA fusion protein carrier in preparation of a polysaccharide-protein conjugate vaccine.

In a fourth aspect, the invention provides a multivalent meningococcal conjugate vaccine in which meningococcal capsular polysaccharides from groups a, C, Y and W135 are conjugated to a recombinant Δ fHbp-NadA fusion protein carrier.

In a fifth aspect, the invention provides a multivalent pneumococcal conjugate vaccine in which pneumococcal capsular polysaccharide is conjugated to a recombinant Δ fHbp-NadA fusion protein carrier.

The invention has the beneficial effects that: 1. aiming at the problem that the capsular polysaccharide component of the group B meningococcus is not suitable for being used as a vaccine, the fusion product delta fHbp-NadA of the recombinant protein of the group B meningococcus human factor H binding protein and the neisseria adhesin A protein is expressed by using a reverse genetics technology. The fusion protein has a Δ fHbp comprising conserved domains of fHbp a and B subfamilies (three variants V1-V3), covering all Nm B group strains expressing fHbp proteins, and a Δ fHbp comprising the complete V of fHbp_A～V_EFive domains whose structure, function and antigenic site maintain integrity; at the same time, NadA of the fusion protein is expressed in 50% of the Nm group B strains, and these strains are essentially highly pathogenic. Therefore, even if the strain expressing NadA is not expressed by fHbp, the strain can be covered by the delta fHbp-NadA protein, and the protein vaccine prepared by using the recombinant protein as the antigen can widely protect almost all Nm B group pathogenic bacteria from infection.

2. The recombinant delta fHbp-NadA is a brand-new carrier protein which can be used for conjugate vaccines, is different from the commonly used protein carriers (CRM197, TT, OMP, DT and HiD) in the existing conjugate vaccine process, and therefore, the immunogenicity of the conjugate vaccine cannot be reduced due to the fact that the same protein carrier is inoculated too much.

3. Although the components of the delta fHbp and the NadA in the delta fHbp-NadA are important factors for the survival and the pathogenesis of Nm, the components are non-toxic, so that the active protein does not need to be detoxified in industrial production, the structure and the antigen active site are not changed, and a better immune effect is obtained.

4. The delta fHbp-NadA fuses and expresses two active proteins by designing a soft connecting peptide chain, and then the two active proteins become a single protein while keeping respective activity, so that the method has more controllability in industrial production and immunization, greatly reduces the risk, and can provide more polysaccharide-protein binding sites.

5. The novel multivalent meningococcal conjugate vaccine taking the recombinant delta fHbp-NadA as the protein carrier has a broad-spectrum immune effect, immune mouse serum can generate a cross-linking reaction with model strains of A, B, C, Y and W135 groups, high pathogenic bacteria which express fHbp (3 variants) in a group B and express NadA but do not express fHbp can be covered, and the broad-spectrum and extremely strong bactericidal effect is shown in bactericidal detection.

6. The novel 23-valent pneumococcal conjugate vaccine taking the recombinant delta fHbp-NadA as the protein carrier has a broad-spectrum immune effect, immune mouse serum can generate a cross-linking reaction with

serotype

1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F Spn strain polysaccharide antigens, and can cover

popular serotype

1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 33F strains in China, and show a broad-spectrum and extremely strong bactericidal effect in bactericidal detection, so that the polysaccharide-protein conjugate vaccine is more suitable for people in China to use.

7. The novel conjugate vaccine taking the recombinant delta fHbp-NadA as the protein carrier has the general advantages of the polysaccharide-protein conjugate vaccine: after immunization, T cell effect can be activated, and immunological memory is generated, so that the infant below 2 years old is really protected.

8. The invention discloses a multivalent capsular polysaccharide-delta fHbp-NadA protein conjugate vaccine prepared by a CDAP activated polysaccharide post-derivatization combination process. Not only eliminates the toxicity of cyanogen bromide in the conventional method, but also obviously improves the activation efficiency.

Drawings

FIG. 1 shows the variable domains and recombinant Δ fHbp domains of fHbp V1-V3.

FIG. 2 shows the amplified electropherogram of Δ fHbp and the electropherogram for identifying pET- Δ fHbp.

FIG. 3 shows the electrophoresis pattern of NadA amplification and pET- Δ fhbp-NadA identification.

FIG. 4 shows IPTG induced expression of Δ fHbp and Δ fHbp-NadA protein in recombinant bacteria.

FIG. 5 identifies Δ fHbp and Δ fHbp-NadA recombinant proteins for Western-Blot.

FIG. 6 is an SDS-PAGE identification of purified Δ fHbp and Δ fHbp-NadA recombinant proteins.

FIG. 7 is SDS-PAGE identifying Δ fHbp-NadA recombinant protein after large scale purification.

Detailed Description

The invention provides a delta fHbp-NadA protein vector and multivalent meningococcal and pneumococcal conjugate vaccines based on the delta fHbp-NadA protein vector, which are further described in the following with reference to specific examples. The following examples are illustrative only and are not to be construed as limiting the invention.

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 delta fHbp and delta fHbp-NadA recombinant proteins

1. Construction of Pet- Δ fHbp and Pet- Δ fHbp-NadA recombinant plasmids

fHbp is a membrane surface lipoprotein, and many meningococcal strains carry their genes, and strains that do not carry them are also found. Meningococcal fHbp can be divided into 3 antigen variant groups V1-V3, based on antigenic cross-reactivity and sequence similarity of the entire protein. Typically, antibodies made against fHbp of variant V1 (also referred to as subfamily B) have bactericidal activity against strains expressing fHbp from variant V1, but not against strains expressing fHbp in variants V2 and V3 (also referred to as subfamily a), and vice versa. The protein molecules of fHbp contain different combinations of five variable domains (V)_A～V_E) Each variable segment is derived from subfamily a and subfamily B. We designed the structure of Δ fHbp (as shown in FIG. 1) based on its domain features, which includes the A, B domain of V1 (with the B domain includingHaving an epitope of V1), and the C, D, E domain of V3 (C, D, E domain contains epitopes of V2 and V3, and the amino acids at position 174 and 216 are highly conserved, and only have individual amino acid differences), theoretically, recombinant Δ fHbp can cover all antigenic sites of wild-type fHbp 3 variants, and has broad-spectrum antibacterial potential.

Based on GeneBank accession full-length CDS sequences of fHbp V1 and fHbp V3, we designed 2 primer pairs as follows: primers were introduced at positions P1 and P4 with BamHI and HindIII sites, respectively.

P1：GGATTCATGAACCGAACTGCCTTCTGCTGCC

P2：GCCGGGCAGTTGGTTGAAGGCGGTA

P3：ACGGCATTCGGTTCAGACGATGCCA

P4：AAGCTTTTACTGCTTGGCGGCAAGACCGATA

PCR amplification was performed using two strains of MenB group bacteria expressing fHbp V1 and V3 as templates (purchased from ATCC, USA). Wherein P1, P2 can amplify A, B domain gene fragment of fHbp V1, and P3 and P4 can amplify C, D, E domain gene fragment of V3. And then the two segments of gene fragments are used as templates, and P1 and P4 are used as primers to carry out bypass PCR, so that the recombinant delta fHbp full-length fragment can be obtained through amplification. Target gene (PCR product) and prokaryotic expression vector pET32a (+) were double digested with BamH I and Hind III, and Δ fhbp gene fragment and linear pET32a vector were recovered from the gel recovery cassette. After the gel is recovered, DNA ligase is used for connecting at 16 ℃ overnight, an escherichia coli DH5a strain is transformed, monoclonal amplification is carried out, after plasmid extraction, a band with the length of about 800bp identified by BamH I and Hind III double enzyme digestion is carried out, positive clones are screened under kanamycin resistance, a recombinant plasmid pET-delta fHbp is obtained, and a PCR amplification electrophoresis pattern and an accurate identification electrophoresis pattern are shown in figure 2. And (3) storing the recombinant plasmid with correct identification at the temperature of-70 ℃ in the form of 15% glycerol bacteria, sequencing and identifying the recombinant plasmid with correct identification, wherein the amino acid sequence of delta fHbp is shown as the sequence table SEQ ID NO: 1, and the nucleotide coding sequence is shown in a sequence table SEQ ID NO: 2(826 bp).

SEQ ID NO：2(826bp)：

ATGAACCGAACTGCCTTCTGCTGCCTTTTCCTGACCACCGCCCTGATTCTGACCGCCTGCAGCAGCGGAGGCGGCGGAAGCGGAAGCGGCGGTGTCGCCGCCGACATCGGCACGGGGCTTGCCGATGCACTAACTACGCCGCTCGACCATAAAGACAAAGGTTTGAAATCTCTGACATTGGAAGACTCCATTCCCCAAAACGGAACACTAACCCTGTCGGCACAAGGTGCGGAAAAAACTTTCAAAGCCGGCGACAAAGACAACAGCCTCAACACGGGCAAACTGAAGAACGACAAAATCAGCCGCTTCGACTTCGTGCAAAAAATCGAAGTGGACGGACAAACCATCACGCTGGCAAGCGGCGAATTTCAAATATACAAACAGGACCACTCCGCCGTCGTTGCCCTACAGATTGAAAAAATCAACAACCCCGACAAAATCGACAGCCTGATAAACCAACGCTCCTTCCTTGTCAGCGGTTTGGGCGGAGAACATACCGCCTTCAACCAACTGCCCGGCACGGCATTCGGTTCAGACGATGCCAGTGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGACACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTTGACCTGGCCGCCTCCGATATCAAGCCGGATAAAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCTCTAGGCATCTTTGGCGGGCAAGCCCAGGAAGTTGCCGGCAGCGCAGAAGTGGAAACCGCAAACGGCATACGCCATATCGGTCTTGCCGCCAAGCAGTAA

The amino acid sequence is SEQ ID NO: 1:

MNRTAFCCLFLTTALILTACSSGGGGSGSGGVAADIGTGLADALTTPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPGTAFGSDDASGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAASDIKPDKKRHAVISGSVLYNQAEKGSYSLGIFGGQAQEVAGSAEVETANGIRHIGLAAKQ

however, there is a drawback to using Δ fHbp protein alone because in some MenB strains the expression level of fHbp protein is low and a vaccine with only Δ fHbp antigen may not be sufficient for a broad range of immunising meningococcal infections. Neisserial adhesin a (nada), binds epithelial cell adhesin/invasin in vitro. This antigen is very conserved (> 96% amino acid identity) in MenB strains, absent in strains of certain genetic lineages, only 50% of MenB-pathogenic strains express NadA protein, but 50% are highly pathogenic. The fusion expression of the delta fHbp protein and the NadA protein can certainly improve the antigenicity of the delta fHbp protein and the NadA protein, but a flexible linker peptide segment is added between the two proteins, namely a Huston designed and synthesized (GGGGS)3 sequence, which is most classical, and is beneficial to the correct folding of the two proteins, so that the respective immune prototypes are not influenced. Based on NadA full-length CDS sequence registered in GeneBank, primers were designed: HindIII and XhoI cleavage sites are respectively introduced into primers P5 and P6, and a sequence of an expression (GGGGS)3 connexin is designed behind the cleavage site of P5.

P5：AAGCTTGGCGGCGGCGGCAGT GGCGGCGGCGGCAGT

GGCGGCGGCGGCAGT ATGAAACACTTTCCATCCAAAGTAC(SEQ ID NO：3)

P6：CTCGAGTTACCACTCGTAATTGACGCCGACA

Using MenB group bacteria expressing NadA as a template (purchased from ATCC in USA), P5 and P6 as primers, a full-length CDS sequence of NadA protein is obtained through amplification, a target gene (NadA product) and a recombinant plasmid pET-delta fHbp are subjected to double enzyme digestion by Hind III and Xho I, and a gel recovery box is used for recovering a NadA gene fragment and a linear pET-delta fHbp vector. After the gel is recovered, DNA ligase is used for connecting at 16 ℃ overnight, an escherichia coli DH5a strain is transformed, monoclonal amplification is carried out, after plasmid extraction, a band of about 1900bp identified by BamH I and Xho I double enzyme digestion is carried out, positive clones are screened under kanamycin resistance, a recombinant plasmid pET-delta fHbp-NadA is obtained, and a PCR amplification electrophoresis pattern and an accurate identification electrophoresis pattern are shown in figure 3. The recombinant plasmid with correct identification is stored at the temperature of-70 ℃ in the form of 15% glycerol bacteria, sequencing and identification are correct, and the amino acid sequence of the recombinant delta fHbp-NadA fusion protein carrier is shown as the sequence table SEQ ID NO: 4, the nucleotide coding sequence is shown in a sequence table SEQ ID NO: 5(1089 bp).

SEQ ID NO：5(1089bp)：

ATGAACCGAACTGCCTTCTGCTGCCTTTTCCTGACCACCGCCCTGATTCTGACCGCCTGCAGCAGCGGAGGCGGCGGAAGCGGAAGCGGCGGTGTCGCCGCCGACATCGGCACGGGGCTTGCCGATGCACTAACTACGCCGCTCGACCATAAAGACAAAGGTTTGAAATCTCTGACATTGGAAGACTCCATTCCCCAAAACGGAACACTAACCCTGTCGGCACAAGGTGCGGAAAAAACTTTCAAAGCCGGCGACAAAGACAACAGCCTCAACACGGGCAAACTGAAGAACGACAAAATCAGCCGCTTCGACTTCGTGCAAAAAATCGAAGTGGACGGACAAACCATCACGCTGGCAAGCGGCGAATTTCAAATATACAAACAGGACCACTCCGCCGTCGTTGCCCTACAGATTGAAAAAATCAACAACCCCGACAAAATCGACAGCCTGATAAACCAACGCTCCTTCCTTGTCAGCGGTTTGGGCGGAGAACATACCGCCTTCAACCAACTGCCCGGCACGGCATTCGGTTCAGACGATGCCAGTGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGACACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTTGACCTGGCCGCCTCCGATATCAAGCCGGATAAAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCTCTAGGCATCTTTGGCGGGCAAGCCCAGGAAGTTGCCGGCAGCGCAGAAGTGGAAACCGCAAACGGCATACGCCATATCGGTCTTGCCGCCAAGCAGTAAGGCGGCGGCGGCAGT GGCGGCGGCGGCAGT GGCGGCGGCGGCAGTATGAAACACTTTCCATCCAAAGTACTGACCACAGCCATCCTTGCCACTTTCTGTAGCGGCGCACTGGCAGCCACAAGCGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGTTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGGTGAAGACGGCACAATTACCCAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGAAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGCTGATATTGCTAAAAACTCAGCACGCATCGACAGCTTGGACAAAAACGTAGCTAATCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGCCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGTAA

The amino acid sequence is SEQ ID NO: 4:

MNRTAFCCLFLTTALILTACSSGGGGSGSGGVAADIGTGLADALTTPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPGTAFGSDDASGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAASDIKPDKKRHAVISGSVLYNQAEKGSYSLGIFGGQAQEVAGSAEVETANGIRHIGLAAKQGGGGS GGGGS GGGGSMKHFPSKVLTTAILATFCSGALAATSDDDVKKAATVAIVAAYNNGQEINGFKAGETIYDIGEDGTITQKDATAADVEADDFKGLGLKKVVTNLTKTVNENKQNVDAKVKAAESEIEKLTTKLADTDAALADTDAALDETTNALNKLGENITTFAEETKTNIVKIDEKLEAVADTVDKHAEAFNDIADSLDETNTKADEAVKTANEAKQTAEETKQNVDAKVKAAETAAGKAEAAAGTANTAADKAEAVAAKVTDIKADIATNKADIAKNSARIDSLDKNVANLRKETRQGLAEQAALSGLFQPYNVGRFNVTAAVGGYKSESAVAIGTGFRFTENFAAKAGVAVGTSSGSSAAYHVGVNYEW

2. prokaryotic expression and identification of delta fHbp and delta fHbp-NadA recombinant proteins

Recombinant plasmids pET-delta fHbp and pET-delta fHbp-NadA are converted into E.coli BL21(DE3), cultured overnight (containing 50 mu g/ml kanamycin) at 37 ℃ to obtain delta fHbp/BL21(DE3) and delta fHbp-NadA/BL21(DE3) expression bacteria, single clones are picked, cultured with shaking at 37 ℃ until the density of the bacteria reaches OD600 of about 0.5-1.0, isopropyl-beta-D-thiogalactoside (IPTG) with the final concentration of 0.5mM is added, cultured with shaking at 37 ℃ for 2-6 hours, and identified by SDS-PAGE, and the results are shown in figure 4, wherein the expression bands are obvious after induction of the delta fHbp/BL21(DE3) and the delta fHbp-NadA/BL21(DE3), and the molecular weights are respectively 30kD and 72 kD. Identified as Δ fHbp and Δ fHbp-NadA recombinant proteins by Western-Blot, as shown in figure 5:

lanes

1 and 3 are Δ fHbp/BL21(DE3) inducible expression product,

lanes

2 and 4 are Δ fHbp-NadA/BL21(DE3) inducible expression product,

lanes

1 and 2 are detected by fHbp monoclonal antibody and have expected bands of 30kD and 72kD respectively,

lanes

3 and 4 are detected by NadA monoclonal antibody, and only lane 4 has a band of 72kD, and the result is in accordance with the expected result. The last two recombinant proteins were identified by mass spectrometry as Δ fHbp and Δ fHbp-NadA recombinant proteins.

Two, small-scale purification of Δ fHbp and Δ fHbp-NadA recombinant proteins and evaluation of their immune effects

Delta fHbp/BL21(DE3) and delta fHbp-NadA/BL21(DE3) were inoculated in LB liquid medium (containing 50. mu.g/ml kanamycin), cultured at 37 ℃ and 200rpm for 12 hours, expanded-cultured at a 1% inoculation ratio, cultured at 37 ℃ and 200rpm for 3 to 6 hours, and then induced at OD600 to 16 to 20 with IPTG (0.5mM) for 4 hours. And centrifuging by a centrifuge to collect the thalli. Sonication, and prior SDS-PAGE showed that recombinant protein was expressed as inclusion bodies (FIG. 3). After washing the inclusion body with TE +300mM NaCl and TE + 1% Triton-100, centrifuging at 8000rpm for 20min to obtain the inclusion body, after washing, dissolving in 3mol/L urea, 20mmol/L LTris-Cl, 1mmol/L EDTA, pH4.0 solution, purifying by CM column ion exchange chromatography, two target proteins with the same size with the target protein can be obtained. And finally, after the recombinant protein is diluted and renatured for 24 hours, quickly concentrating the recombinant protein at a low temperature by an ultrafilter to 1.2 times of the original volume, passing through an anion exchange column Q Sepharose F.F, and collecting a target protein peak, wherein the detection method is as shown in the figure 4 and the figure 5, the content of the gel detection protein reaches over 95.3 percent, the purification effect is as shown in the figure 6, and WB detection is confirmed to be the target protein. After desalting by dialysis against PBS (pH7.4), the protein concentration was determined to be about 0.5mg/mL by BCA.

Mouse immunization: respectively using purified delta fHbp and delta fHbp-NadA recombinant protein to subcutaneously immunize 10 SPF-level NIH mice with the age of 6 weeks, wherein the immunization dose is 50 ug/purified delta fHbp or delta fHbp-NadA recombinant protein (dissolved in 0.2ml of physiological saline), the immunization program is 0, 2 and 4 weeks, blood is collected 2 weeks after the immunization is finished, and serum is centrifugally collected; another 10 mouse controls were set and saline was injected in the same manner. The collected serum is used for measuring the titer of the serum antibody by an ELISA method, and the specific method is as follows: respectively coating an enzyme label plate with a proper dose by using purified delta fHbp or delta fHbp-NadA recombinant protein, incubating overnight at 37 ℃, washing the plate, adding serial diluted mouse serum to be detected, incubating at 37 ℃, washing the plate, adding horseradish peroxidase labeled goat anti-mouse IgG secondary antibody for color development, measuring by using an enzyme label reader, and reading an absorbance value (A value) of 492nm (630nm is a reference wavelength). The standard deviation of the negative control group serum A mean value plus 3 times is the Cutoff value, the serum A value to be detected is greater than the Cutoff value, the positive result is judged, and the geometric mean titer of each type of mice is calculated by the maximum dilution that the A value is greater than the Cutoff value, namely the mouse serum IgG antibody titer (Table 1). Both recombinant proteins produced high and low levels of serum antibodies upon immunization of mice.

TABLE 1 measurement of serum antibody titers (geometric mean) after three immunizations of mice with each group of antigens

The whole thallus coating selects MenB group strains MC58 (high expression fHbp V1 variant), 8047 (high expression fHbp V2 variant), M1239 high expression fHbp V3 variant), 961-⁸The enzyme-linked immunosorbent assay plate is coated with 96-well enzyme-linked immunosorbent assay plate per ml at the concentration, 100 ul/well, coated overnight at 4 ℃, and detected after the plate is washed, and the result is shown in table 2. Also, MenB group strains MC58 (high expression fHbp V1 variant), 8047 (high expression)fHbp V2 mutable), M1239 high expression fHbp V3 mutable), 961-5945 (medium expression fHbp protein) and 67/00 (low expression fHbp protein), followed by bactericidal assay with the corresponding serum antibodies, and bactericidal antibody titers were determined and calculated (the highest serum dilution at 50% or more bactericidal rate compared to complement negative control wells was the serum antibody bactericidal titer), with the results shown in table 3.

TABLE 2 Whole cell ELISA for each MenB Strain

TABLE 3 bactericidal activity of each MenB strain experiment BC50titer (1:)

Remarking: bactericidal power test with a ratio of greater than 1: 8 is protective.

As can be seen from the combined analysis of the results in tables 2 and 3, the Δ fHbp protein vaccine was able to effectively immunize against infection with all MenB strains expressing fHbp (V1, V3 and V2 variants), but was unable to protect against MenB strains that express low or even no fHbp; the delta fHbp-NadA protein vaccine can immunize all MenB strains expressing fHbp (V1, V3 and V2 variants) and has protection effect on MenB strains with low expression even without expression of fHbp. Meanwhile, the delta fHbp-NadA protein vaccine can generate higher antibody titer and has stronger bactericidal capacity, which shows that the fusion expression of the two specific proteins can play a role in synergistically promoting immune response and can provide a broader-spectrum vaccine protection function. Therefore, our subsequent process selects Δ fHbp-NadA recombinant proteins as protein carriers for novel multivalent meningococcal and pneumococcal conjugate vaccines.

Thirdly, fermentation culture, induced expression and large-scale antigen protein concentration and purification of engineering bacteria

And preparing the recombinant strain with the correctly identified delta fHbp-NadA/BL21(DE3) into an original seed bank and a working seed bank according to the requirements of pharmacopeia. The process flow is as follows:

1. taking engineering strain work seed bank strains, scratching an LB agar culture medium (containing kanamycin), culturing overnight at 37 ℃, selecting a single colony, inoculating the single colony into an LB liquid culture medium (containing 50 mu g/ml of kanamycin), gradually expanding the single colony to a fermentation tank for fermentation culture according to the inoculation proportion of 1-2%, when OD600 reaches 16-20, adopting isopropyl-beta-D-thiogalactoside (IPTG) for induction culture for 4-8 hours, stopping culture, and centrifugally collecting thalli by a large-capacity centrifuge.

2. Adding bacteria breaking buffer (50mM PB, 2mM EDTA, pH7.5), homogenizing at high pressure (800-1000bar) for two times, centrifuging (10000rpm, 60min, 8 deg.C) to obtain supernatant. Fractionated precipitation with 30-60% ammonium sulphate, 50mM PBPH7.5 lysis, 50kD ultrafiltration 5 times and concentration to 1/5-1/3 in bulk.

3. Sepharose QFF chromatography was used, equilibration solution: 50mM PB pH7.2, eluent: 50mM PB +1M NaClPH7.2. The elution method comprises the following steps: gradient eluting with 0-100%, and collecting 10% eluate.

4. And (3) performing ultrafiltration concentration on the 10% gradient eluate by using a 50KD ultrafiltration membrane pack, performing Sepharose4FF gel filtration chromatography, collecting a protein peak at V0, performing ultrafiltration for more than 5 times by using 0.15mol/L sodium chloride and 10KD, concentrating until the protein content is 1-2mg/ml, and performing aseptic filtration with the particle size of 0.22 mu m. The delta fHbp-NadA protein stock solution is obtained by aseptic examination, protein content examination, molecular weight and purity examination, protein specificity examination and bacterial endotoxin examination.

The technical requirement is to obtain a recombinant delta fHbp-NadA protein stock solution with the purity of more than 95%. The bacterial endotoxin is not higher than 20EU/ml, and the invention should be not higher than 10EU/ml, and optimally not higher than 5 EU/ml. Molecular weight and purity checks are shown in FIG. 7, with a purity of 96.3%. Protein specific detection WB detection was the target protein as shown in fig. 5.

Preparation of four-multivalent pneumococcus-delta fHbp-NadA conjugate vaccine and immune effect evaluation

The recombinant delta fHbp-NadA is prepared by a biological engineering technology, is a brand-new carrier protein which can be used for conjugate vaccines, is different from the common protein carriers (CRM197, TT, OMP, DT and HiD) in the existing conjugate vaccine process, and therefore, the immunogenicity of the conjugate vaccine cannot be reduced due to excessive inoculation of the same protein carrier. Meanwhile, although the delta fHbp and the NadA are important factors for Nm survival and pathogenesis, the delta fHbp and the NadA are non-toxic, so that detoxification treatment on the active protein is not needed in industrial production, the structure and the antigen active site are not changed, and a better immune effect is obtained. And thirdly, the delta fHbp-NadA fuses and expresses the two active proteins through a flexible connecting peptide chain, and the two active proteins become a single protein while keeping respective activity, so that the method has higher controllability and greatly reduced risk during industrial production and immunization, and can provide more polysaccharide-protein binding sites. Therefore, the delta fHbp-NadA can be used as a novel high-quality carrier protein for developing conjugate vaccines of other pathogenic bacteria (such as Hib and Spn), like the membrane protein OMP of Nm and the bacterial surface protein HiD of Hib, and can be completely used as a conventional carrier protein. The invention provides a strong immunogenic pneumococcal conjugate vaccine capable of covering 23 serotypes by using delta fHbp-NadA recombinant protein as a carrier. The preparation of the delta fHbp-NadA recombinant protein stock solution is as described above, and the subsequent specific preparation scheme is as follows:

1.23 serotype Spn mycopoda polysaccharide preparation

Selecting 23 serotypes (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F) of pneumococcus by fermentation culture, separating culture solution by adopting a sand culture centrifuge, a disc centrifuge or other high-capacity centrifuges, and collecting centrifugal supernatant; ultrafiltering the supernatant with 100KD membrane, concentrating, precipitating with 25-80% ethanol, collecting precipitate, and washing with anhydrous ethanol and acetone to obtain crude polysaccharide; dissolving polysaccharide in sterile water for injection, treating with sodium deoxycholate, refining crude sugar by series chromatography or fractional chromatography using GE filler Capto adhere and Capto DEAE or ion exchange filler with the same functional property of other manufacturers, collecting flow-through peak, desalting the flow-through peak by GE Sephadex G25Coarse, precipitating with ethanol or lyophilizing to recover polysaccharide, and storing at-20 deg.C.

Preparation of 2.23-valent Spn bacterial polysaccharide-delta fHbp-NadA protein conjugate vaccine

The multivalent capsular polysaccharide-delta fHbp-NadA protein conjugate vaccine is also prepared by applying a CDAP activated polysaccharide post-derivatization binding process. The specific method comprises the following steps: dissolving 23 serotype Spn bacterial polysaccharides to 5-15mg/ml, adjusting pH to about 10.8, adding 1/10(g/g) CDAP into the polysaccharide solution, and activating the polysaccharides for 8min at room temperature under the alkaline environment of maintaining pH at 10.8. According to the following steps: 1 (volume ratio to activated polysaccharide) and adding 0.5mol/L of adipic dihydrazide to react for 50-70min at room temperature. Removing cyanogen bromide and dihydrazide by 50KD membrane ultrafiltration to obtain polysaccharide derivative. The polysaccharide derivative was mixed with the carrier protein Δ fHbp-NadA at a ratio of 1: 1(g/g) and stirred in the following ratio of carbodiimide: polysaccharide 1: 10, adding carbodiimide, and reacting for 60min at room temperature in an acidic environment (pH5.6). Removing impurities by ultrafiltration with 100KD ultrafiltration membrane, and concentrating. Finally, gel chromatography purification was performed using GE Sepharose4FF, and the eluate near V0 was collected. And (3) sterilizing and filtering the mixture by using a 0.22 mu m filter membrane to obtain a 23-valent Spn bacterial polysaccharide-delta fHbp-NadA protein conjugate vaccine stock solution.

The stock solution can be stirred and adsorbed by adopting an aluminum phosphate adjuvant, diluted by 0.15mol/L sodium chloride for preparation, the final concentration of the multivalent glycoprotein conjugate is respectively 20 mu g/ml in terms of polysaccharide content, the final concentration of the aluminum content is 0.45-0.6mg/ml, the mixture is uniformly stirred, and the mixture is subpackaged by a prefilled syringe and 0.5 ml/branch to prepare a 23-valent Spn bacterial polysaccharide-delta fHbp-NadA protein conjugate vaccine preparation which is stored at the temperature of 2-8 ℃. Or adding 80mg/ml sucrose as excipient into the vaccine stock solution, lyophilizing each polysaccharide content to obtain protein vaccine preparation, and storing at 2-8 deg.C.

Evaluation of Effect of 3.23-valent Spn bacterial polysaccharide-delta fHbp-NadA protein conjugate vaccine

Mouse immunization: 10 SPF (specific pathogen free) level NIH (human immunodeficiency virus) mice with 6 weeks of age are immunized subcutaneously by using 23-valent Spn bacterial polysaccharide-delta fHbp-NadA protein conjugate vaccine protein stock solution and a commercial 23-valent positive control vaccine preparation, the immunization dose is 0.2 ml/mouse at a time, the immunization program is 0, 2 and 4 weeks, blood is collected 2 weeks after the immunization is finished, and serum is collected centrifugally; another 10 mouse controls were set and saline was injected in the same manner. The collected serum is used for measuring the titer of the serum antibody by an ELISA method, and the specific method is as follows: respectively coating an enzyme label plate with a proper dose of purified 23 kinds of serotype polysaccharides, incubating overnight at 37 ℃, washing the plate, adding serial diluted mouse serum to be detected, incubating at 37 ℃, washing the plate, adding horseradish peroxidase labeled goat anti-mouse IgG secondary antibody for color development, measuring by an enzyme label instrument, and reading the absorbance value (A value) of 492nm (630nm is reference wavelength). The standard deviation of the negative control group serum A mean value plus 3 times is the Cutoff value, the serum A value to be detected is greater than the Cutoff value, the positive result is judged, and the geometric mean titer of each type of mice is calculated by the maximum dilution that the A value is greater than the Cutoff value, namely the mouse serum IgG antibody titer (Table 4). After the conjugate vaccine protein stock solution and a commercial 23-valent positive control polysaccharide vaccine are used for immunizing mice, high and low serum antibodies are generated, and the titer generated by the conjugate vaccine protein stock solution is higher, which indicates that the protein antigen has the function of synergistically promoting immunity.

TABLE 4 titer determination (geometric mean) of the antibodies of the various serotypes after three immunizations of the vaccine on the mice

The immune effect of conjugate vaccine and polysaccharide vaccine is evaluated by two methods, whole cell ELISA and bactericidal activity test of epidemic strain are measured by whole cell coating,

serotype

1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 33F of highly pathogenic Spn strain popular in China are selected for whole cell coating, after the strains are expanded, cultured and proliferated, bacterial lawn is scraped, dissolved by normal saline, sterilized by formaldehyde and diluted to 2 × 10⁸Coating 96-well enzyme label plate with the concentration, 100 ul/well, coating overnight at 4 ℃, washing the plate, and performing ELISA detection by using corresponding vaccine serum antibody, wherein the result is shown in Table 5.

TABLE 5 Whole cell ELISA for each serotype pneumococcal strain

Similarly, highly pathogenic Spn strains

serotype

1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 33F were cultured and then bactericidal detection was performed using the corresponding vaccine serum antibodies, and bactericidal antibody titers were determined and calculated (the highest serum dilution at a bactericidal rate of 50% or more compared to the complement negative control wells was the serum antibody bactericidal titer), with the results shown in table 6.

TABLE 6 Sterilization test for various serotype pneumococcal strains BC50titer (1:)

The results are comprehensively analyzed, and the novel 23-valent pneumococcal conjugate vaccine taking the recombinant delta fHbp-NadA as the protein carrier has broad-spectrum immune effect, the serum of an immunized mouse can generate cross-linking reaction with polysaccharide antigens of

serotype

1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F Spn strains, meanwhile, the strain can cover the

serotype

1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 33F strains which are popular in China, and shows broad-spectrum and extremely strong bactericidal effect in bactericidal detection, therefore, people (especially infants, old and weak people) are protected from pneumonia caused by invasion of most pneumococcal pathogenic bacteria, and the polysaccharide-protein conjugate vaccine is more suitable for people in China.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Wuhan Bowo Biotechnology Ltd

<120> polyvalent pneumococcal conjugate vaccine and preparation method thereof

<130>2017

<160>5

<170>PatentIn version 3.3

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Met Asn Arg Thr Ala Phe Cys Cys Leu Phe Leu Thr Thr Ala Leu Ile

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Leu Thr Ala Cys Ser Ser Gly Gly Gly Gly Ser Gly Ser Gly Gly Val

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Asp His Lys Asp Lys Gly Leu Lys Ser Leu Thr Leu Glu Asp Ser Ile

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Pro Gln Asn Gly Thr Leu Thr Leu Ser Ala Gln Gly Ala Glu Lys Thr

65 70 75 80

Phe Lys Ala Gly Asp Lys Asp Asn Ser Leu Asn Thr Gly Lys Leu Lys

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Asn Asp Lys Ile Ser Arg Phe Asp Phe Val Gln Lys Ile Glu Val Asp

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Gly Gln Thr Ile Thr Leu Ala Ser Gly Glu Phe Gln Ile Tyr Lys Gln

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Asp His Ser Ala Val Val Ala Leu Gln Ile Glu Lys Ile Asn Asn Pro

130 135 140

Asp Lys Ile Asp Ser Leu Ile Asn Gln Arg Ser Phe Leu Val Ser Gly

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Leu Gly Gly Glu His Thr Ala Phe Asn Gln Leu Pro Gly Thr Ala Phe

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Gly Ser Asp Asp Ala Ser Gly Lys Leu Thr Tyr Thr Ile Asp Phe Ala

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Ala Lys Gln Gly His Gly Lys Ile Glu His Leu Lys Ser Pro Glu Leu

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Asn Val Asp Leu Ala Ala Ser Asp Ile Lys Pro Asp Lys Lys Arg His

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Lys Gln

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atgaaccgaa ctgccttctg ctgccttttc ctgaccaccg ccctgattct gaccgcctgc 60

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ggcgaatttc aaatatacaa acaggaccac tccgccgtcg ttgccctaca gattgaaaaa 420

atcaacaacc ccgacaaaat cgacagcctg ataaaccaac gctccttcct tgtcagcggt 480

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gccagtggaa aactgaccta caccatagat ttcgccgcca agcagggaca cggcaaaatc 600

gaacatttga aatcgccaga actcaatgtt gacctggccg cctccgatat caagccggat660

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accgcaaacg gcatacgcca tatcggtctt gccgccaagc agtaa 825

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aagcttggcg gcggcggcag tggcggcggc ggcagtggcg gcggcggcag tatgaaacac 60

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Met Asn Arg Thr Ala Phe Cys Cys Leu Phe Leu Thr Thr Ala Leu Ile

1 5 10 15

Leu Thr Ala Cys Ser Ser Gly Gly Gly Gly Ser Gly Ser Gly Gly Val

20 25 30

Ala Ala Asp Ile Gly Thr Gly Leu Ala Asp Ala Leu Thr Thr Pro Leu

35 40 45

Asp His Lys Asp Lys Gly Leu Lys Ser Leu Thr Leu Glu Asp Ser Ile

50 55 60

Pro Gln Asn Gly Thr Leu Thr LeuSer Ala Gln Gly Ala Glu Lys Thr

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Phe Lys Ala Gly Asp Lys Asp Asn Ser Leu Asn Thr Gly Lys Leu Lys

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Asn Asp Lys Ile Ser Arg Phe Asp Phe Val Gln Lys Ile Glu Val Asp

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Gly Gln Thr Ile Thr Leu Ala Ser Gly Glu Phe Gln Ile Tyr Lys Gln

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Asp His Ser Ala Val Val Ala Leu Gln Ile Glu Lys Ile Asn Asn Pro

130 135 140

Asp Lys Ile Asp Ser Leu Ile Asn Gln Arg Ser Phe Leu Val Ser Gly

145 150 155 160

Leu Gly Gly Glu His Thr Ala Phe Asn Gln Leu Pro Gly Thr Ala Phe

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Gly Ser Asp Asp Ala Ser Gly Lys Leu Thr Tyr Thr Ile Asp Phe Ala

180 185 190

Ala Lys Gln Gly His Gly Lys Ile Glu His Leu Lys Ser Pro Glu Leu

195 200 205

Asn Val Asp Leu Ala Ala Ser Asp Ile Lys Pro Asp Lys Lys Arg His

210 215 220

Ala Val Ile Ser Gly Ser Val Leu Tyr Asn Gln Ala Glu Lys Gly Ser

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Tyr Ser Leu Gly Ile Phe Gly Gly Gln Ala Gln Glu Val Ala Gly Ser

245 250 255

Ala Glu Val Glu Thr Ala Asn Gly Ile Arg His Ile Gly Leu Ala Ala

260 265 270

Lys Gln Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

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Ser Met Lys His Phe Pro Ser Lys Val Leu Thr Thr Ala Ile Leu Ala

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Thr Phe Cys Ser Gly Ala Leu Ala Ala Thr Ser Asp Asp Asp Val Lys

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Lys Ala Ala Thr Val Ala Ile Val Ala Ala Tyr Asn Asn Gly Gln Glu

325 330 335

Ile Asn Gly Phe Lys Ala Gly Glu Thr Ile Tyr Asp Ile Gly Glu Asp

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Gly Thr Ile Thr Gln Lys Asp Ala Thr Ala Ala Asp Val Glu Ala Asp

355 360 365

Asp Phe Lys Gly Leu Gly Leu Lys Lys Val Val Thr Asn Leu Thr Lys

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Thr Val Asn Glu Asn Lys Gln Asn Val Asp Ala LysVal Lys Ala Ala

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Glu Ser Glu Ile Glu Lys Leu Thr Thr Lys Leu Ala Asp Thr Asp Ala

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Ala Leu Ala Asp Thr Asp Ala Ala Leu Asp Glu Thr Thr Asn Ala Leu

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Asn Lys Leu Gly Glu Asn Ile Thr Thr Phe Ala Glu Glu Thr Lys Thr

435 440 445

Asn Ile Val Lys Ile Asp Glu Lys Leu Glu Ala Val Ala Asp Thr Val

450 455 460

Asp Lys His Ala Glu Ala Phe Asn Asp Ile Ala Asp Ser Leu Asp Glu

465 470 475 480

Thr Asn Thr Lys Ala Asp Glu Ala Val Lys Thr Ala Asn Glu Ala Lys

485 490 495

Gln Thr Ala Glu Glu Thr Lys Gln Asn Val Asp Ala Lys Val Lys Ala

500 505 510

Ala Glu Thr Ala Ala Gly Lys Ala Glu Ala Ala Ala Gly Thr Ala Asn

515 520 525

Thr Ala Ala Asp Lys Ala Glu Ala Val Ala Ala Lys Val Thr Asp Ile

530 535 540

Lys Ala Asp Ile Ala Thr Asn Lys Ala Asp Ile Ala Lys AsnSer Ala

545 550 555 560

Arg Ile Asp Ser Leu Asp Lys Asn Val Ala Asn Leu Arg Lys Glu Thr

565 570 575

Arg Gln Gly Leu Ala Glu Gln Ala Ala Leu Ser Gly Leu Phe Gln Pro

580 585 590

Tyr Asn Val Gly Arg Phe Asn Val Thr Ala Ala Val Gly Gly Tyr Lys

595 600 605

Ser Glu Ser Ala Val Ala Ile Gly Thr Gly Phe Arg Phe Thr Glu Asn

610 615 620

Phe Ala Ala Lys Ala Gly Val Ala Val Gly Thr Ser Ser Gly Ser Ser

625 630 635 640

Ala Ala Tyr His Val Gly Val Asn Tyr Glu Trp

645 650

<210>5

<211>1959

<212>DNA

<213> Artificial Synthesis

<400>5

atgaaccgaa ctgccttctg ctgccttttc ctgaccaccg ccctgattct gaccgcctgc 60

agcagcggag gcggcggaag cggaagcggc ggtgtcgccg ccgacatcgg cacggggctt 120

gccgatgcac taactacgcc gctcgaccat aaagacaaag gtttgaaatc tctgacattg 180

gaagactcca ttccccaaaa cggaacacta accctgtcgg cacaaggtgc ggaaaaaact 240

ttcaaagccg gcgacaaaga caacagcctc aacacgggca aactgaagaa cgacaaaatc 300

agccgcttcg acttcgtgca aaaaatcgaa gtggacggac aaaccatcac gctggcaagc 360

ggcgaatttc aaatatacaa acaggaccac tccgccgtcg ttgccctaca gattgaaaaa 420

atcaacaacc ccgacaaaat cgacagcctg ataaaccaac gctccttcct tgtcagcggt 480

ttgggcggag aacataccgc cttcaaccaa ctgcccggca cggcattcgg ttcagacgat 540

gccagtggaa aactgaccta caccatagat ttcgccgcca agcagggaca cggcaaaatc 600

gaacatttga aatcgccaga actcaatgtt gacctggccg cctccgatat caagccggat 660

aaaaaacgcc atgccgtcat cagcggttcc gtcctttaca accaagccga gaaaggcagt 720

tactctctag gcatctttgg cgggcaagcc caggaagttg ccggcagcgc agaagtggaa 780

accgcaaacg gcatacgcca tatcggtctt gccgccaagc agtaaggcgg cggcggcagt 840

ggcggcggcg gcagtggcgg cggcggcagt atgaaacact ttccatccaa agtactgacc 900

acagccatcc ttgccacttt ctgtagcggc gcactggcag ccacaagcga cgacgatgtt 960

aaaaaagctg ccactgtggc cattgttgct gcctacaaca atggccaaga aatcaacggt 1020

ttcaaagctg gagagaccat ctacgacatt ggtgaagacg gcacaattac ccaaaaagac 1080

gcaactgcag ccgatgttga agccgacgac tttaaaggtc tgggtctgaa aaaagtcgtg 1140

actaacctga ccaaaaccgt caatgaaaac aaacaaaacg tcgatgccaa agtaaaagct 1200

gcagaatctg aaatagaaaa gttaacaacc aagttagcag acactgatgc cgctttagca 1260

gatactgatg ccgctctgga tgaaaccacc aacgccttga ataaattggg agaaaatata 1320

acgacatttg ctgaagagac taagacaaat atcgtaaaaa ttgatgaaaa attagaagcc 1380

gtggctgata ccgtcgacaa gcatgccgaa gcattcaacg atatcgccga ttcattggat 1440

gaaaccaaca ctaaggcaga cgaagccgtc aaaaccgcca atgaagccaa acagacggcc 1500

gaagaaacca aacaaaacgt cgatgccaaa gtaaaagctg cagaaactgc agcaggcaaa 1560

gccgaagctg ccgctggcac agctaatact gcagccgaca aggccgaagc tgtcgctgca 1620

aaagttaccg acatcaaagc tgatatcgct acgaacaaag ctgatattgc taaaaactca 1680

gcacgcatcg acagcttgga caaaaacgta gctaatctgc gcaaagaaac ccgccaaggc 1740

cttgcagaac aagccgcgct ctccggcctg ttccaacctt acaacgtggg tcggttcaat 1800

gtaacggctg cagtcggcgg ctacaaatcc gaatcggcag tcgccatcgg taccggcttc 1860

cgctttaccg aaaactttgc cgccaaagca ggcgtggcag tcggcacttc gtccggttct 1920

tccgcagcct accatgtcgg cgtcaattac gagtggtaa 1959

Claims

1. A multivalent pneumococcal conjugate vaccine characterized by: the recombinant delta fHbp-NadA fusion protein carrier comprises recombinant delta fHbp, a flexible connecting peptide section and NadA, and the recombinant delta fHbp comprises V of fHbp variant V1_A、V_BDomains and V of fHbp variant V3_C、V_D、V_EAnd the amino acid sequence of the recombinant delta fHbp is shown in a sequence table SEQ ID NO: 1, nucleotide coding thereofThe code sequence is shown in a sequence table SEQ ID NO: 2, the nucleotide coding sequence of the flexible connecting peptide segment is shown as a sequence table SEQ ID NO: 3, the amino acid sequence of the recombinant delta fHbp-NadA fusion protein vector is shown in a sequence table SEQ ID NO: 4, the nucleotide coding sequence is shown in a sequence table SEQ ID NO: 5, the pneumococcal capsular polysaccharide is capsular polysaccharide on the capsule of the separated and purified serotype pneumococcus, and the serotypes of the serotype pneumococcus are 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.

2. The method of preparing a multivalent pneumococcal conjugate vaccine of claim 1, wherein: the method comprises the following steps:

s1, preparing recombinant delta fHbp-NadA protein;

s2, preparing pneumococcal capsular polysaccharide;

s3, preparing the multivalent capsular polysaccharide-delta fHbp-NadA protein conjugate vaccine by using CDAP activated polysaccharide post-derivatization combination.

3. The method of claim 2, wherein the step of S1 of preparing the recombinant Δ fHbp-NadA protein fusion protein vector further comprises the step of

S1a, designing primers to respectively amplify to obtain a fHbp V1 gene fragment and a fHbp V3 gene fragment, performing bypass PCR amplification by taking the two gene fragments as templates to obtain a recombinant delta fHbp full-length fragment, recovering the delta fHbp gene fragment and a pET vector, and preparing a recombinant plasmid pET-delta fHbp;

s1b, designing a full-length CDS sequence of the NadA protein through primer amplification, recovering a NadA gene fragment and a recombinant plasmid pET-delta fHbp, and preparing the recombinant plasmid pET-delta fHbp-NadA;

s1c, converting the recombinant plasmid pET-delta fHbp-NadA into an expression strain to obtain the recombinant delta fHbp-NadA fusion protein vector.