WO2020259559A1

WO2020259559A1 - Varicella-zoster virus ge protein mutant and expression method therefor

Info

Publication number: WO2020259559A1
Application number: PCT/CN2020/098019
Authority: WO
Inventors: 周晨亮; 何强; 沈巧英; 周凌云; 江元翔; 刘革; 曾宪放; 史力; 莫呈钧; 张智
Original assignee: 怡道生物科技(苏州)有限公司
Priority date: 2019-06-28
Filing date: 2020-06-24
Publication date: 2020-12-30
Also published as: CN112142829A; CN112142829B

Abstract

Provided are an immunogenic varicella-zoster virus gE protein mutant and a coding gene thereof. Compared to a wild-type varicella-zoster virus gE protein, the mutant differs in that leucine is mutated into methionine at position 141 of a mature antigen sequence. Further provided is a preparation method for the mutant.

Description

Varicella-zoster virus gE protein mutant and its expression method

Technical field

The invention belongs to the field of biomedicine. Specifically, the invention relates to a recombinant shingles vaccine composition and a preparation method and application thereof.

Background technique

Varicella zoster virus (VZV) is one of the eight human herpes viruses, also called type 3 human herpes viruses. Varicella-zoster virus is spread all over the world and is highly contagious. So far, only one serotype has been found. In nature, VZV only infects humans. It can cause chickenpox and herpes zoster (HZ). Chickenpox is usually seen in childhood, while herpes zoster does not develop until adulthood. After the primary infection with chickenpox, the virus can be latent in the host's ganglia. With age, immune function impairment or immunosuppression, VZV can be reactivated and cause shingles. The clinical manifestation of herpes zoster is a unilateral vesicular rash. The obvious feature is that it is limited to a single skin segment, usually accompanied by radicular pain. Patients may have obvious pain and discomfort. Symptoms can last for several weeks or months. In severely ill patients, they can even last for several years, resulting in a decline in the quality of life. In rare cases, shingles may not appear as a rash. Complications occur in about 25% of people with shingles, and they increase with age. The most common serious complication is post-herpetic neuralgia (PHN), that is, pain that persists after the acute phase of herpes. The incidence of herpes zoster patients is 10%-30%. The pain can be It lasts for several months or even years, seriously affecting the quality of life of patients. The risk factors that affect the onset of shingles are age, immune deficiency, gender, and other potential factors.

Most primary VZV infections occur in childhood, then VZV is latent in the ganglia, and VZV can be reactivated in adulthood. Studies have shown that about 99% of Americans aged 40 and over have serological evidence of VZV infection; 90% of Europeans aged 20-29 years are seropositive for anti-VZV; in some countries in South America, Australia and Asia The primary VZV infection may occur later, but 90% of people over 40 years old will have VZV seropositivity. Therefore, on a global scale, most adults are at risk of developing shingles and its related complications. The global incidence of herpes zoster is (3～5)/1000 person-years, and the Asia-Pacific region is (3～10)/1000 person-years, and it is increasing by 2.5%～5.0% year by year, and the hospitalization rate is (2～25)/100,000 The mortality rate is (0.017～0.465) per 100,000 person-years, and the recurrence rate is 1%～6%. At present, my country is in a state of high-degree aging, and the social and economic burden brought by HZ is increasing year by year. For individuals, HZ has brought a huge negative impact on the quality of life of patients, especially elderly patients. According to data released by the National Bureau of Statistics, it is estimated that the population over 40 years old in 2017 was about 650 million. If the HZ incidence rate is 2.5/1000 person-years, it is conservatively estimated that there are about 1.6 million new cases of HZ in my country each year.

Since medication can only relieve symptoms, vaccination is the best strategy to prevent HZ and its complications. Currently, only two HZ vaccines are on the market worldwide (HZ live attenuated vaccine Zostavax and HZ subunit vaccine Shingrix). Merck’s Zostavax is a live attenuated vaccine. It contains the same virus strain as the VZV Oka strain used in the varicella vaccine. The vaccine formula uses a minimum potency of 19400 PFU. It was approved by the FDA in 2006 for marketing and has been approved in more than 60 countries. One dose can be subcutaneously administered to people over 50 years of age. Shingrix developed by GlaxoSmithKline is a subunit vaccine based on recombinant gE protein supplemented with a novel adjuvant AS01B. Phase III clinical trial data show that this subunit vaccine has better immunogenicity and effectiveness than Zostavax in the elderly. , And was approved by the FDA in 2017 to be marketed. It is suitable for people aged 50 and above and requires 2 doses. At present, Shingrix has been conditionally approved for marketing in China, but domestic vaccines under development are all attenuated live vaccines, and their protective efficacy and immunity durability will be lower than Shingrix. Shingrix is out of stock due to production capacity and other issues, so there is an urgent need for a domestic vaccine The self-developed subunit vaccine helps reduce the burden of disease caused by shingles and its complications.

Although some vaccines against VZV have been developed in the prior art, there are still problems such as low expression efficiency of VZV gE protein, low activity of the expressed protein, and unsatisfactory immune effect. Therefore, there is a need to develop improved VZV vaccine products in this field.

Summary of the invention

The technical problem to be solved by the present invention is to provide a new VZV gE protein mutant as a candidate antigen for preventing herpes zoster. Another problem to be solved by the present invention is to provide an efficient and economical method for expressing VZV gE protein mutants in CHO cells.

The first aspect of the present invention provides a VZV gE protein mutant, which is different from the amino acid sequence of the VZV gE protein included in the NCBI (National Center for Biotechnology Information) database.

There have been quite a lot of studies on VZV gE protein: wild-type or full-length gE protein is generally 623 amino acids. It consists of the main part of the gE protein containing the signal peptide, the hydrophobic anchor region (residues 546-558) and the C-terminal tail (for example, refer to the literature Davison AJ, Scott J: The complete DNA sequence of varicella-zoster virus. J Gen Virol 1986; 67: 1759-1816). Different studies have slightly different differences in the protein molecular structure of gE protein. Some researchers divide protein molecules into hydrophilic extracellular domain (including signal peptide), hydrophobic transmembrane domain (residues 545-561) and intracellular tail ( For example, refer to Grose C. Glycoproteins encoded by varicella-zoster virus: biosynthesis, phosphorylation and intracellular trafficking. Annu Rev Microbil. 1990, 44: 59-80). However, those skilled in the art can understand that the above-mentioned different distinguishing methods do not make a substantial difference in the application and preparation of gE protein. The protein in a typical pharmaceutical composition will be different from the full-length protein, but a truncated protein. For example, when using modern biomolecular technology to prepare recombinant VZV gE protein, the gE protein is generally removed (truncated) so that it lacks the carboxy-terminal hydrophobic anchor region (for example, patent CN107106675A, EP0405867B); or called transmembrane region (hydrophobic). Region) and intracellular region (for example, patent CN102517302A). At the same time, when the mutant protein precursor is translated using the translation mechanism in the carrier cell (host cell) and transferred to the cell membrane to be secreted outside the cell, the signal peptide region is usually cut by signal peptidase (for example, patent CN102548578A, CN102711812B). Therefore, typically, a mutant that can be effectively applied to a recombinant shingles vaccine composition should have a mutation site in the epitope region, that is, in the extracellular region that does not contain a signal peptide (for convenience of description, in the present invention Called mature antigen in Chinese). It should be noted that, as a conventional technical means in this field, the anchor region, transmembrane region, intracellular region and signal peptide can be predicted by related application software, such as SignalP (http://www.cbs.dtu. dk/services/SignalP/) predict whether the protein has a signal peptide, and use TMHMM Server V.2.0 (http://www.cbs.dtu.dk/services/TMHMM) to predict the transmembrane region and intracellular region of the protein , Using PSORT software can determine the accuracy of the secreted signal peptide and whether the signal peptide cleavage site can be recognized and cleaved.

In the present invention, the mutation site of the VZV gE protein mutant is at position 141 of the mature antigen sequence (region), and this site is mutated from leucine to methionine. It should be noted that the VZV gE protein sequence included in the NCBI database indicates that position 141 of the mature antigen region is highly conserved. For example, NCBI Sequence ID=Q9J3M8.1 amino acid sequence (full length is 623 amino acids), based on the amino acid sequence, according to the content disclosed in the prior art EP0405867B (appropriate VZV gE antigen is truncated to remove the carboxyl end The VZV glycoprotein gE (also called gpl) in the anchor region (starting at amino acid 547), and the protein sequence generally obtained by expressing proteins in eukaryotic cells will lack the leader sequence (also called signal peptide, positions 1-30) Amino acid), it can be deduced that the mature antigen in the full-length protein is 31-546 positions, and the 141st position is leucine. Similarly, if a similar protein analysis is performed on the amino acid sequences of Sequence IDs AQT34120.1, AGY33616.1, and AEW88548.1 in the NCBI database, the result is that the 141st position of the mature antigen region in these proteins is leucine.

The inventor of the present application surprisingly discovered that when selecting a specific amino acid sequence as the basis for gene optimization in the process of preparing recombinant gE protein, if the 141th position of the mature antigen region is artificially modified (mutation from leucine to methionine) , Genes and vectors designed based on the modified amino acid sequence will be able to achieve higher antigen expression. As an example, the ideal VZV gE protein used in the preparation of recombinant shingles vaccine composition can be as shown in SEQ ID NO: 1, where the sequence 1-30 is the signal peptide region, and the 31-546 is mature In order to increase the expression level of the protein, compared with the wild-type VZV gE protein, the inventors artificially mutated position 141 of the mature antigen region from leucine to methionine.

The second aspect of the present invention also provides a recombinant shingles vaccine composition, which contains a gE protein of the VZV virus. In a preferred embodiment, the aforementioned gE protein has an amino acid sequence shown in SEQ ID NO: 1 or 3.

The production of VZV gE protein is usually achieved through expression in cultured cells or through chemical synthesis. Host cells frequently used and suitable for protein production include E. coli, yeast, insects, and mammals. Expression vectors and host cells are commercially available, and the expression vector contains a promoter and a cloning site for the sequence encoding the protein of interest so that the promoter and sequence are operably linked. Other elements may be present, such as a signal peptide sequence (sometimes called a leader sequence), a tag sequence (e.g., hexa-His), a transcription termination signal, an origin of replication, and a sequence encoding the product. The methods and procedures used to transfect host cells are also well known. As mentioned earlier, a suitable VZV gE antigen is a VZV gE protein that has been truncated to remove the carboxy-terminal anchor region (starting at amino acid 547) (for example, patent CN107106675A, EP0405867B). At the same time, the expression of protein in eukaryotic cells will generally make the obtained protein sequence lack the signal peptide part (for example, patent CN102548578A, CN102711812B). Therefore, in a typical embodiment, when CHO cells are used to express gE protein, the sequence of gE protein that is finally secreted outside the CHO cell will be as shown in SEQ ID NO: 3. However, it should be noted that various techniques can also be used to inhibit the cleavage of the aforementioned signal peptide during cell production. For example, one or more amino acids formed at the cleavage site differ from amino acids, so that the sequence is not recognized or cleaved by the cell. Therefore, in a typical example of obtaining protein by inhibiting signal peptide cleavage, the final gE protein obtained will be shown in SEQ ID NO:1. As mentioned above, according to the data in NCBI, the mature antigen region is highly conserved among various gE wild-type or full-length proteins. Therefore, those skilled in the art can understand that the mutants provided by the present invention are not limited to The above two specific sequences can be obtained by those skilled in the art through software prediction methods known in the art and published prior art content. These sequences have similarities with SEQ ID NO:1, namely There is a mutation in the 141th of the mature antigen region (from leucine to methionine). Due to the different sources of VZV virus strains, the difference between these sequences may be 1 or 2 or 3 or 4 amino acids The difference in residues, but these differences will not affect the application of gE protein as an antigen.

In the second aspect of the present invention, a gE gene encoding the gE protein of the VZV virus is provided, the gene is a gE gene that can be expressed in CHO cells, and the gene has the nucleoside shown in SEQ ID NO: 2 Acid sequence.

In the third aspect of the present invention, an expression vector is provided, and the expression vector contains the sequence of the gE gene.

In a preferred embodiment, the expression vector is cloned to carry the blasticidin resistance gene by introducing the 5'and 3'ends of the gE gene into the restriction endonuclease sites of XbaI and NotI, respectively. The plasmid expression vector and the plasmid expression vector carrying the bleomycin resistance gene.

In a preferred embodiment, the expression vector contains the aforementioned gE gene shown in SEQ ID NO: 2.

In the fourth aspect of the present invention, a genetically engineered cell is provided, the cell contains the expression vector, or the gE gene shown in SEQ ID NO: 2 is integrated in its genome.

In a preferred embodiment, the aforementioned cells are CHO cells.

In a preferred embodiment, the cell is a CHO cell containing the gE gene or expression vector of the present invention, and the cell can express and produce gE protein at a high level.

In the fifth aspect of the present invention, there is provided an immunogenic protein, the protein is the gE protein of the VZV virus, and the gE protein is expressed by CHO cells.

In a preferred embodiment, the immunogenic protein is prepared by the following method:

(1) Culturing the genetically engineered cells to express the gE protein of the VZV virus in the cells;

(2) Isolate the gE protein of the VZV virus.

In the sixth aspect of the present invention, the use of the vaccine composition is provided to prevent or treat diseases or disorders related to herpes zoster virus infection.

In the seventh aspect of the present invention, a method for expressing the gE protein of VZV virus in CHO cells is provided, which includes the following steps:

(1) Cloning the gE gene of the present invention into an expression vector;

(2) Transform the expression vector obtained in step (1) into CHO cells;

(3) Obtain cell lines stably expressing gE protein through the screening of mini cell populations and monoclonal screening;

(4) Use the cell line obtained in step (3) for expression to obtain VZV protein.

According to a specific embodiment of the present invention, the expression vector described in the step (1) is a plasmid expression vector carrying a blasticidin resistance gene and/or a plasmid expression vector carrying a bleomycin resistance gene .

According to a specific embodiment of the present invention, the CHO cells used in the step (2) are CHO-K1 cells.

Other aspects of the present invention are obvious to those skilled in the art due to the disclosure herein.

Detailed ways

Example 1 Cloning, expression and purification of varicella-zoster virus gE protein

1. Selection of gE protein and (gE gene) synthesis

Through NCBI database and literature search, a conservative truncated gE protein amino acid sequence was selected as the basis for gene optimization. In order to improve the expression efficiency, only the signal peptide region and mature antigen were selected. At the same time, the inventor of the present application surprisingly found that when selecting a specific amino acid sequence as the basis for gene optimization in the process of preparing recombinant gE protein, if the 141th position of the mature antigen region is artificially modified (mutated from leucine to methionine) Acid), genes and vectors designed based on the modified amino acid sequence will be able to achieve higher antigen expression. The mutated protein sequence has a total length of 546 amino acids (SEQ ID NO: 1), which is specifically as follows. The sequence includes the signal peptide and the main body of the antigen, but does not include the C-terminal carboxy-terminal anchoring region in the gE protein.

In order to facilitate the efficient expression of gE protein in CHO cells, the codon preferred by CHO cells is selected to optimize the gE gene (such as SEQ ID NO: 2), and the synthesis is entrusted to an outsourcing company, as follows. It should be noted that the optimization principle is not simply to select the most frequent codon in CHO cells, but a more complex optimization scheme. There are three overall optimization principles: First, according to the degeneracy of the codons, replace the original codons with the high-frequency codons corresponding to each amino acid in CHO cells; second, to avoid excessive GC in the post-transcribed mRNA The content affects its secondary structure, which in turn affects translation efficiency. In the optimization process, try to control the GC% of the gene at 40-60%; third, avoid some commonly used restriction enzyme sites.

2. Cloning and construction of gE protein expression plasmid

The 5'and 3'ends of the above synthesized gE gene were introduced into Xba I and Not I restriction endonuclease cleavage sites, respectively, and the fragment was cloned into the expression vectors pWX2.0 and pWX1.0 by PCR. The vector pWX2.0 carries the blasticidin resistance gene, and pWX1.0 carries the bleomycin resistance gene. Both of these vectors use the cytomegalovirus (CMV) promoter/enhancer sequence to express the target gene. The CMV promoter is a strong promoter commonly used to promote eukaryotic gene expression. The corresponding expression plasmid was obtained by cloning and construction, and the sequence was verified by restriction enzyme digestion and sequencing.

It should be noted that the construction methods of the above two expression vectors are conventional technical means in the field. As an example, the construction methods that can be referred to are:

2.1 Construction of expression vector pWX2.0-B-gE

Using plasmid pUC57-gE as a template, double digestion with Xba I (NEB, Cat.#: R0145S) and Not I-HF (NEB, Cat.#: R3189S) to obtain a 1660 bp DNA fragment, 1% agarose gel After separation by electrophoresis, the gel was tapped under UV light and the 1660bp DNA fragment was recovered.

The vector pWX2.0 was digested with restriction enzymes Xba I (NEB, Cat. #: R0145S) and Not I-HF (NEB, Cat. #: R3189S), and the digested product with a size of 4775 bp was recovered. The above-mentioned 1660bp recovered fragment was ligated into the above 4775bp pWX2.0 vector, and the ligated product was transformed into TOP10 competent cells, and screened with a plate containing blasticidin to obtain several monoclonal positive colonies, and some of them were selected for PCR Amplification and sequencing verification. Then, pick a clone that was verified by sequencing and streak it twice on the LB plate. Transfer the isolated single clone to 300ml LB medium (containing 100μg/mL ampicillin), culture it with shaking overnight at 37°C and 220rpm , A large amount of plasmid DNA was extracted, and the finally obtained plasmid was named pWX2.0-B-gE.

2.2 Construction of expression vector pWX1.0-Z-gE

Same as step 2.1, using plasmid pUC57-gE as template, double enzyme digestion with Xba I and Not I-HF to obtain a 1660 bp DNA fragment. After separation by 1% agarose gel electrophoresis, the gel was tapped and recovered under UV light. 1660bp DNA fragment.

The vector pWX1.0 was double digested with restriction enzymes Xba I and Not I-HF, and a 4172 bp vector fragment was recovered using a gel purification kit. The 1660bp recovered fragment was ligated to the 4172bp pWX1.0 vector fragment, and the ligated product was transformed into TOP10 competent cells. Several single clones were selected and verified by PCR and sequencing. Then, pick a clone that is correct after sequencing verification, streak it twice on the LB plate, transfer the isolated single clone to 300ml LB medium (containing 100μg/mL ampicillin sodium, you can also choose such as Bola (Mycin), 37°C, 220rpm overnight shaking culture, a large amount of plasmid DNA extraction, the final plasmid named pWX1.0-Z-gE.

3. Expression and purification of gE protein expression plasmid

The expression plasmid was prepared in large quantities by the method in 2 above, and linearized and stably transfected into host cell CHO-K1. In this example, a total of 6 sets of transfection experiments were performed. Subsequently, each group of transfected mini cell populations were screened by fed-batch culture, and the cell populations with higher expression levels were selected for subsequent clonal screening by limiting dilution. It can be seen that in all 6 groups of mini cell groups, the average cell expression level is between 2-3g/L, and the expression level of the highest three groups of cells is between 2.5-3g/L, the highest group of cells The protein expression level can be 3.04g/L. On this basis, the three groups of cell populations with the highest expression levels (respectively from different plasmids, but the cell growth is better) are selected, and single clones are selected for these three groups of cell populations by limiting dilution. The selected single clones are expanded during the fed-batch culture process, the supernatants of the above cloned cells are collected, and the samples are taken for western blot detection. The target protein is determined according to the bands, and the cloned LDC photos, growth conditions, and expression levels are considered. , Viable cell density, viability, final lactic acid content and related product quality parameters, select the best three clones, that is, the dominant cell line. The cell line obtained above is cultured and expressed in a bioreactor to obtain a cell culture supernatant containing gE protein. Similarly, the above-mentioned supernatant can be sampled for Western blot detection to determine whether it is the target protein according to the band. It has been proved that the average gE protein in the above cell lines can reach 3g/L. It should be noted that if the wild-type protein sequence is used (as a comparative example, the wild-type protein sequence of NCBI Sequence ID=Q9J3M8.1, but also does not include the C-terminal carboxy-terminal anchor region in the gE protein) is used as the preparation of recombinant gE protein The design template is optimized by the same DNA optimization strategy, and the average protein expression of the obtained cell line is about 2.5g/L. In addition, the mutant provided by the present invention has immunogenicity equivalent to that of the wild-type protein (detailed below). Therefore, the protein mutants, expression vectors and cell lines containing optimized nucleic acid sequences provided by the present invention will provide a more excellent alternative to the VZV vaccine. At the same time, the amino acid sequence analysis of the expression product proved that the protein precursor (that is, as the gene When the optimized basic amino acid sequence is secreted outside the cell, the signal peptide region has been cut by the signal peptidase. At this time, the VZV gE recombinant protein is shown in SEQ ID NO: 3, and there is an artificial mutation at position 141 of the sequence. The amino acid was mutated to methionine.

It should be noted that the method of using CHO cell lines to stably express the VZV gE recombinant protein is a well-known method in the art. For details, please refer to the "Molecular Cloning Test Guide" and other documents, such as Haumont M, et al., Virus Research 40 (1996 ), 199-204, Purification, characterization and immunogenicity of recombinant varicella-zoster virus glycoprotein gE secreted by Chinese hamster ovary cells. As an example, the specific plasmid transfection and cell line construction methods are as follows:

Use CD CHO medium M1 to resuscitate and cultivate 1 CHO-K1 host cell as a working cell bank cell.

The plasmids pWX1.0-Z-gE and pWX2.0-B-gE obtained by the above 2 method were linearized, specifically, the restriction enzyme Sca I-HF was used for digestion (50μl digestion system ), take 2μl of the digested product and use 1% agarose gel electrophoresis to detect it. The results show that the two plasmids after Sca I-HF digestion showed a single and clear band, indicating a good linearization result. The 50 μl linearized product was purified by phenol chloroform extraction and ethanol precipitation, and then dissolved in 10 mM Tris buffer. By Nano-Drop detection, the concentration of plasmid pWX1.0-Z-gE was 1285.3ng/μl, and the concentration of pWX2.0-B-gE was 1064.3ng/μl. Subsequently, the above host cell CHO-K1 was inoculated into the medium M1 at a density of 7×10 ⁵ cells/ml. After 24 hours, the host cells were counted, and the cells were diluted to 1.0×10 ⁶ cells/ml with the pre-warmed medium M1, and then 5ml of the cell suspension was placed in a Kuhner shaker for later use. The parameters of the shaker are: temperature 36.5°C, humidity 85%, carbon dioxide 6%, rotating speed 225rpm. Prepare for transfection, the specific steps are as follows:

First, take 12μg each of the plasmid pWX1.0-Z-gE and plasmid pWX2.0-Z-gE, and add them to a 50ml shake tube pre-added with 776μl OptiProSFM. At the same time, add 24μl of FreeStyle Max Reagent to another 50ml shaker tube pre-filled with 776μL of OptiProSFM. Afterwards, add the mixture of FreeStyle Max Reagent and OptiProSFM to the mixture of plasmid and OptiProSFM, gently pipette to mix and incubate at room temperature for 10 minutes;

Second, take 667 μl of the above mixed solution (plasmid, FreeStyle Max Reagent and OptiProSFM) and drop it into the diluted host cell suspension (5 ml). The cells were then incubated in a Kuhner shaker. The parameters of the shaker are: temperature 36.5°C, humidity 85%, carbon dioxide 6%, speed 225rpm;

Third, after incubating for 6 hours, add 5ml of pre-warmed fresh medium M1. The cells were then placed in a Kuhner shaker for culture. The parameters of the shaker are: temperature 36.5°C, humidity 85%, carbon dioxide 6%, rotating speed 225rpm.

The stable CHO-K1 cell line containing the optimized gE gene can be obtained by the above-mentioned transfection method. At the same time, media selection of mini cell populations and selection of monoclonal cells by the limiting dilution method are also well-known experimental methods. Through these methods, monoclonal cells with higher expression levels can be obtained, that is, the dominant CHO cell line.

Example 2 Evaluation of immunogenicity of recombinant shingles vaccine

After obtaining the gE protein described in Example 1, after conventional processing methods such as hydrophobic chromatography, anion exchange chromatography, hydroxyapatite chromatography, ultrafiltration, and nanofiltration, the protein with a purity of 95% or more can be obtained Used as an antigen protein.

In order to study the immunogenicity of the antigens prepared by the genes and vectors provided by the present invention, the above antigens and adjuvants were sucked into a recombinant shingles vaccine composition, and C57BL/6 mice were used as animal models to carry out immunization. Original research. The specific method is: using gE protein as an antigen, using aluminum phosphate and CpG ODN as adjuvants to prepare a recombinant shingles vaccine composition. C57BL/6 mice aged 6-8 weeks were selected to be randomly divided into groups, 10 mice in each group, the above vaccine composition was injected intramuscularly, and the vaccine group and adjuvant group were set up, 0 and 3 weeks were immunized, and the blood was collected in the 5th week. The ELISA method was used to detect the antibody titer (ie total IgG) of the anti-VZV gE protein in the serum, and the ELISPOT method was used to detect the cellular immunity level in the spleen cells, mainly the expression of IFN-γ. The results show that the vaccine combination prepared by the gE protein obtained by the technical solution provided by the present invention has very good immunogenicity and can be used as a potential recombinant shingles vaccine candidate (specific results are shown in Table 1).

The immunogenicity evaluation method is a professional technical method in the field. As an example, the more specific operation method is as follows:

1. Animal experiment of recombinant shingles vaccine

C57BL/6 mice aged 6-8 weeks were selected to be randomly divided into groups, with 10 mice in each group. Different doses of vaccine were injected intramuscularly (see Table 1 for the specific ratio) with an injection volume of 0.05ml; 0 and 3 weeks of immunization, blood was taken from the spleen at the 5th week, serum was separated for ELISA detection of antibodies, and splenic lymphocytes were separated for ELISPOT analysis. The specific detection method can be taken as an example as follows:

2. Antibody titer detection

(1) The antigen gE stock solution was diluted with PBS to 1μg/ml, and 100 μl of the diluted stock solution was added to each well of the ELISA plate. 4°C overnight. Washing machine.

(2) Prepare 5% skimmed milk with PBS, and add 100μl of skimmed milk to each well of the ELISA plate. Incubate at 37°C for 2 hours. Washing machine.

(3) PBS is equipped with 2% skimmed milk, the serum to be tested is gradually diluted, and 100 μl of diluted serum is added to each well of the ELISA plate, and incubated at 37°C for 1 hour. Washing machine.

(4) Dilute the goat anti-mouse at a ratio of 1:10000 with 2% skimmed milk prepared in PBS, and add 100 μl of secondary antibody diluted with skimmed milk to each well of the ELISA plate. Incubate at 37°C for 1 hour. Washing machine.

(5) Prepare the chromogenic solution in the proportion of 9ml of chromogenic buffer, 1ml of TMB, and 10μl of 3% H ₂ O ₂ . Add 100μl color developing solution to each well of ELISA plate. Incubate at 37°C for 10 minutes. Add 50μl stop solution to each well of the ELISA plate.

(6) 450nm/620nm reading.

3. Cellular immune detection

Spleens were taken from each group of mice and lymphocytes were separated. The level of IFN-γ expression in mouse spleen lymphocytes was measured by ELISPOT.

(1) Coated ELISPOT plate (aseptic operation, performed one day before taking the spleen)

Wet the ELISPOT plate with 35% alcohol, add 15μl/well to the 96-well ELISPOT plate, and the retention time does not exceed 1 minute. Add 200μl/well sterile water to wash the plate 5 times. Take 150μl of IFN-γ coated antibody, add it to 10ml PBS, mix well, and filter with 0.2μm filter. Take the coating antibody diluent, add 100μl/well to 96-well ELISPOT plate, 4℃ overnight.

(2) ELISPOT plate sealing (sterile operation)

Discard the coated antibody, and wash the plate 5 times by adding 200μl/well of sterile PBS. Add the 1640 complete medium (containing 10% FBS) to the 96-well ELISPOT plate at a volume of 200 μl/well, and block at room temperature for more than 30 minutes. Discard the liquid and buckle dry water on the sterilized gauze to avoid air bubbles when adding the medium in the next step.

(3) Lymphocyte preparation (aseptic operation)

The mice were sacrificed and immersed in 75% ethanol. Take out the mouse spleen in a clean bench. Put a burned 200-mesh copper mesh in a 35mm petri dish, add 1ml of lymphocyte separation solution, and grind with the plunger of a 1ml syringe. After filtering the suspension with spleen cells through a burned 200-mesh copper mesh, transfer to a 15ml centrifuge tube, add the lymphocyte separation solution to 4ml, and cover the surface with 0.5ml of RPMI1640 basic medium. Room temperature, 800g, speed 3, centrifugation for 30 minutes. Aspirate the lymphocyte layer, add 10ml RPMI1640 basal medium, wash, and centrifuge at 250g at room temperature for 10 minutes. Discard the supernatant, add 2ml RPMI1640 complete medium to resuspend the cells, and count.

(4) Sample addition (aseptic operation)

Add cells: Dilute the cells with complete medium to 6×10 ⁶ /ml according to the results of the cell count, and add mAb CD28-A to the cell suspension diluted 1000 times. Add 100μl/well to the ELISPOT plate. Positive control: 1μl ConA stimulant was added, and the stimulation concentration was 5μg/ml. Sample to be tested: add stimulus gE protein peptide library diluted with serum-free medium, final concentration 2μg/ml; negative control: no ConA stimulus, nor stimulus short peptide. Incubate at 37°C with 5% CO2 for 24 hours, during which time the culture plate cannot be moved to avoid changes in the cell position and blur of ELISPOT spots.

(5) Spot detection

Discard the cell suspension, add 200μl/well of sterile PBS and wash the plate 5 times. Take 50 μl of biotin-labeled detection antibody and add it to 10 ml of diluent (PBS+0.1% BSA), mix well, and filter with 0.2 μm filter. Add 100μl to each well and incubate at 37°C for 2 hours. Discard the diluent of the biotin-labeled detection antibody, add 200μl/well of sterile PBS and wash the plate 5 times. Dilute the antibody with diluent (PBS+0.1% BSA), take 50μl and add 10ml of diluent, mix well, and filter with 0.2μm filter. Add 100μl to each well and incubate at 37°C for 1 hour. Start from this step to avoid light operation. Discard the antibody diluent, add 200μl/well of sterile PBS and wash the plate 5 times. Add Fluorescence enhancer-II to a 96-well ELISPOT plate at the amount of 50μl/well, and incubate at 37°C for 15 minutes. Discard the liquid in the plate, buckle the plate upside down on absorbent paper, and pat dry the small drops of water. Remove the protective layer and place it in an electric heating constant temperature incubator, and dry the film at 37°C in the dark. Place the ELISPOT board in

In the S5 VersC CnClyzer enzyme-linked spot image automatic analyzer, adjust the appropriate parameters for spot counting.

The specific results are shown in Table 1 below:

Table 1

All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

A mutant of wild-type varicella-zoster virus gE protein, wherein the mutant is at position 141 of the mature antigen sequence compared with the mature antigen sequence in the wild-type varicella-zoster virus gE protein Mutation from leucine to methionine.
The mutant according to claim 1, wherein the mature antigen sequence is shown in the mature antigen sequence part of SEQ ID NO:1.
A gE gene, which encodes the gE protein of varicella-zoster virus, is characterized in that the gE gene can express the gE protein in CHO cells.
The gE gene according to claim 3, the gene sequence is shown in SEQ ID NO: 2.
An expression vector containing the sequence of the gene of claim 3 or 4.
The expression vector of claim 5, which is a plasmid expression vector carrying a blasticidin resistance gene and/or a plasmid expression vector carrying a bleomycin resistance gene.
A genetically engineered cell containing the expression vector of claim 5, or the gene of claim 3 or 4 integrated in its genome.
The cell of claim 7, wherein the cell is a CHO cell.
An immunogenic protein, the protein is the gE protein of the VZV virus, and the gE protein is expressed by CHO cells.
The immunogenic protein according to claim 9, characterized in that it is shown in SEQ ID NO: 3.
The immunogenic protein according to claim 10, wherein the immunogenic protein is prepared by the following method:

(1) Culturing the genetically engineered cell of claim 7 to express the gE protein of the VZV virus in the cell;

(2) Isolate the gE protein of the VZV virus.
A method for preparing the immunogenic protein of claim 9, characterized in that the method comprises:

(1) Culturing the genetically engineered cell according to claim 7 to express the gE protein of the VZV virus in the cell;

(2) Isolate the gE protein of the VZV virus.
A method for expressing the gE protein of VZV virus in CHO cells includes the following steps:

(1) Cloning the gE gene of the present invention into an expression vector;

(2) Transform the expression vector obtained in step (1) into CHO cells;

(3) Obtain cell lines stably expressing gE protein through the screening of mini cell populations and monoclonal screening;

(4) Use the cell line obtained in step (3) for expression to obtain VZV protein.