WO2021103434A1 - 一种重组水痘带状疱疹病毒疫苗 - Google Patents
一种重组水痘带状疱疹病毒疫苗 Download PDFInfo
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Definitions
- VZV Varicella-zoster virus
- Herpesviridae herpesvirus genus alpha herpesvirus subfamily. It is a double-stranded DNA virus with a diameter of 150 to 200 nm. It is morphologically composed of nucleic acid core and protein. Concentric circular structure formed by capsid and envelope.
- the VZV genome is a linear double-stranded DNA molecule of about 125 kb, which contains a unique long fragment (UL) of about 100 kb and a unique short fragment (US) of about 5.4 kb. They are separated by 6.8 kb terminal and internal repeats, and there are 72 in total.
- Open reading frames in addition to encoding protein molecules related to virus replication, transcription, packaging, release and other biological activities, as well as proteins that interact with host cells, also encode gB, gC, gE, gH, There are 8 glycoproteins, gI, gK, gL and gM, which play an extremely important role in virus maturation and packaging.
- gE glycoprotein E, gE
- ORF68 Open reading frames
- N-terminus is a hydrophilic extracellular region of 544 AA (containing signal peptide), and the C-terminus is a transmembrane hydrophobic region of 17AA and an intracellular region of 62AA.
- gE is a type I membrane protein, which is an essential glycoprotein for the production of infectious VZV particles. It is also the most antigenic and abundant glycoprotein on the viral envelope and the infected cell membrane. At the same time, it is also widely present on the surface of VZV particles. The cell membrane and cytoplasm of the host cell can induce cellular immunity and humoral immunity.
- the glycoprotein E of the VZV virus includes two epitope coding regions e1 and c1. These epitopes are stable among VZV strains from different regions and have high conservation. They are more suitable vaccine antigen candidates. unit. gE exists on the surface of VZV virus particles and on the surface and cytoplasm of infected cells. It exists in the form of different sugar-containing polypeptides at different stages of virus maturity. The gE molecule contains a transport signal and can participate in the assembly of viral proteins and the formation of the envelope in the Golgi apparatus.
- VZV antibodies are mainly directed against the three glycoproteins of gB, gH and gE, especially gE as the main target.
- gE induces specific humoral and cellular immunity produced by the body, which can protect the body from virus attack.
- gE contains phosphorylated high-mannose O-chain and N-chain complex type glycans, which can cooperate with gI to bind to the Fc segment of human IgG.
- gE and gI are covalently linked and act as Fc receptors on the surface of infected cells. The role of.
- Seroepidemiological surveys show that 95%-97% of women of childbearing age are positive for serum anti-VZV antibodies, so the chance of infants becoming infected with varicella virus within 6 months after birth is very low.
- Sero-negative adults small babies born to seronegative pregnant women, immunocompromised persons, fetuses born to pregnant women who have had chickenpox in the fourth to fifth months of pregnancy, and newborns born to mothers who have had chickenpox before and after delivery are all prone to severe Primary infection of VZV.
- the elderly, people with immunodeficiency, children whose mothers had chickenpox during pregnancy, and children who have had chickenpox one year after birth have an increased risk of developing shingles.
- CN102517302A discloses a method for recombinantly expressing truncated glycoprotein E of varicella-zoster virus and its application.
- the varicella-zoster virus (VZV) truncated glycoprotein E (gpE) gene with the transmembrane region and the intracellular region added and His tag added is introduced into the host cell, and the recombinant varicella-zoster virus (gpE) is obtained by expression.
- Herpes virus truncated glycoprotein E helps to increase the expression level of the target protein, simplifies the downstream purification work, and can easily realize the large-scale production of the protein.
- the mRNA of VZV gE was detected by RT-PCR, and the immunoreactivity of gE was detected by western blot and indirect immunofluorescence.
- the expression product was purified by Ni ++ -NTA column and coated on ELISA plate. 127 normal children aged 0-10 The level of VZV-IgG antibody in the serum was detected. Results
- the COS-7 cell line capable of stably expressing the extracellular domain gene of VZVgE was successfully screened.
- the mRNA of gE was detected by RT-PCR. After immunoprinting and indirect immunofluorescence identification, the expressed gE was immunoreactive.
- the specificity of the recombinant protein was identified by SDS-PAGE electrophoresis and western blotting, and the expressed protein was purified and refolded on the column using Ni++-NTA column.
- the eukaryotic plasmid was transfected into COS-7 cells mediated by liposome, the cell line stably expressing VZV gE protein was screened by G418, and the expression product was purified by Ni++ -NTA column; VZV gE gene was detected by RT-PCR MRNA, immunoblotting and indirect immunofluorescence to detect the immunoreactivity of the gE fusion protein.
- Fc receptors are expressed on the surface of many innate immune cells. Therefore, Fc fragments are also widely used in the study of DC targeting.
- Fc receptors are classified into Fc ⁇ R (IgA) according to the different antibody subtypes that the receptor binds to. , Fc ⁇ / ⁇ R (IgA and IgM), Fc ⁇ R (IgE) and Fc ⁇ R (IgG).
- Fc receptors can be divided into high-affinity receptors and low-affinity receptors according to their affinity. High-affinity receptors bind monoclonal antibodies, and low-affinity antibodies bind polyclonal antibodies.
- the application of targeting Fc ⁇ R was the earliest.
- Fc ⁇ R targeting can significantly increase the efficiency of antigen presentation in vitro, promote the binding of antigen to MHC II, and target Fc ⁇ R antigens.
- CD4 + T cells to activate the TH1 signaling pathway (Dai X, Jayapal M, Tay HK, Reghunathan R, et al. Differential signal transduction, membrane trafficking, and immune effector functions mediated by Fc ⁇ RI versus Fc ⁇ RIIa.Blood. 2009; 114 :318-27.).
- the present invention links the extramembrane region encoding the peptide chain of the VZV virus gE protein with the gene encoding the CH2-CH3 region of human immunoglobulin, inserts the target gene into the eukaryotic expression vector, transfects the CHO cell, and successfully expresses it A VZV gE-Fc fusion protein was released.
- the recombinant protein was purified by affinity chromatography, ion exchange chromatography column, molecular sieve chromatography, and virus inactivation to remove possible virus contamination, and a highly purified fusion protein was obtained.
- the present invention relates to a method for preventing and/or reducing the severity of herpes zoster and/or post-herpes zoster neuralgia (PHN), comprising supplying an individual with a combination of live attenuated VZV (OKA strain) or fully inactivated VZV VZV antigen or its recombinant immunogenic derivative gE protein immunogenic composition.
- PPN post-herpes zoster neuralgia
- the present invention also relates to a method for preventing or ameliorating herpes zoster reactivation and/or post-herpetic neuralgia, the method comprising delivering to an individual in need thereof a gE fusion protein or an immunogenic derivative thereof in combination with an adjuvant Or immunogenic fragments of immunogenic compositions or vaccines.
- one of the objectives of the present invention is to provide a varicella-zoster virus glycoprotein E-Fc gene fusion protein to achieve high-purity varicella-zoster virus glycoprotein E The expression product in mammals.
- a recombinant varicella-zoster virus vaccine preparation comprising a fusion protein formed by the amino acid sequence of the extracellular region of the recombinant glycoprotein gE of the live attenuated VZV strain (OKA strain) gene and the Fc segment of human immunoglobulin. Fusion protein, its amino acid sequence is SEQ ID No. 1.
- the vaccine preparation of the present invention also contains a vaccine adjuvant.
- the vaccine adjuvant is selected from: aluminum hydroxide adjuvant, aluminum phosphate adjuvant, or a mixture of aluminum hydroxide and aluminum phosphate adjuvant.
- each dosage unit contains 5 to 200 ⁇ g of the fusion protein.
- the vaccine formulation of the present invention contains 10-100 ⁇ g of the fusion protein in each dosage unit.
- the vaccine formulation of the present invention more preferably, contains 20-60 ⁇ g of the fusion protein in each dosage unit.
- the vaccine preparation of the present invention also contains other substances that can enhance immunogenicity, including but not limited to: phosphatidylcholine, lecithin, 3D-MPL, long-chain fatty acids (esters), mineral oil, vegetable oil, methyl Composition of sodium cellulose, sodium carboxymethyl cellulose, and liposomes containing cholesterol.
- substances that can enhance immunogenicity including but not limited to: phosphatidylcholine, lecithin, 3D-MPL, long-chain fatty acids (esters), mineral oil, vegetable oil, methyl Composition of sodium cellulose, sodium carboxymethyl cellulose, and liposomes containing cholesterol.
- the vaccine preparation of the present invention is a freeze-dried preparation.
- the lyophilized preparation is dissolved by adding aluminum hydroxide adjuvant suspension before use, and after being evenly mixed, it is injected intramuscularly or subcutaneously.
- the present invention further provides a recombinant gene, which can express the fusion protein of the present invention, and its DNA sequence is SEQ ID No.2.
- the fusion protein (hereinafter referred to as gE-Fc or VZV gE) expression vector of the present invention is formed by inserting the gE-Fc fusion gene into the eukaryotic mammalian cell expression vector.
- the fusion protein of the present invention is an exogenous antigen or derivative thereof that can efficiently trigger the immune system of the human body to produce an immune response.
- the immune response refers to the ability to induce the immune system response of the body to produce high titer Serum neutralizing antibodies. Such a response can reduce the incidence of herpes zoster disease in the population, or can alleviate pain or symptoms such as intercostal neuralgia caused by herpes zoster.
- Use common techniques in the art such as serum neutralizing antibody level or VZV gE (glycosylated protein) ELISA antibody level measurement to evaluate the improvement of immune response, or use known clinical criteria to evaluate the improvement in clinical symptoms or signs.
- the above-mentioned expression product can be mixed with aluminum adjuvants or other drugs that enhance immunogenicity to prepare for the prevention of infectious diseases such as intercostal neuralgia caused by herpes zoster in elderly people over 50 years old. It can also be used in infants to prevent infections caused by varicella-zoster virus.
- the protective antigens of VZV also belong to glycoproteins, especially glycoprotein gE, which is the main glycosylated protein of VZV. Its diversified glycosylation forms form the main neutralizing antigen of VZV.
- glycoprotein gE glycoprotein gE
- Existing studies have confirmed that serum antibodies against gE protein can neutralize VZV.
- Suitable antigens also include various glycoproteins, such as gB, gH, gC, gI, IE63 (for example, see Huang et al. J. Virol. 1992, 66: 2664, Sharp et al. J. Inf. Dis. 1992, 165: 852, Debrus, J Virol. 1995 May; 69(5): 3240-5 and references therein), IE62 (for example, see Arvin et al. J. Immunol. 1991 146: 257, Sabella J Virol. 1993 Dec; 67(12): 7673 6 and references therein), ORF4 or ORF 10 (Arvin et al. Viral Immunol. 2002 15:507.), but the abundance of these glycoproteins on the VZV membrane is lower than the abundance of gE, which does not constitute the body’s production of VZV neutralization The main source of antibodies.
- IE63 for example, see Huang et al. J. Virol. 1992, 66: 2664, Sharp
- the present invention realizes the high-efficiency expression of VZV glycoprotein E gene in mammalian expression system. Without changing its amino acid sequence, we synthesize the VZV glycoprotein E extracellular region gene according to the preferred codon optimized codon of CHO cells After constructing the gE-Fc fusion gene, the eukaryotic expression vector was constructed and transfected into CHO K1 cells. Protein A affinity chromatography was used to detect its protein expression, and ELISA was used to detect the specificity of VZV gE and human anti-Varicella virus The binding activity of sex immunoglobulin confirmed the successful secretion and expression of VZV glycoprotein E gene in CHO cells.
- VZV gE immunized rabbits and BALB/C mice The highly purified VZV gE immunized rabbits and BALB/C mice, and the immune serum neutralized the OKA strain virus serum.
- the neutralization test proved that the expressed VZV gE-Fc protein has good immunogenicity. After 2 immunizations Can produce high titer serum neutralizing antibodies.
- the dosage of VZV gE antigen should be a dosage that can induce the body to produce an immune protective response without obvious adverse side effects.
- the amount of antigen used varies with the adjuvant used and the way it exists. Generally speaking, it can be estimated that each dose may contain about 2 ⁇ 1000 ⁇ g of VZV gE; when used in the human body, in order to reduce the number of injections, aluminum adjuvant can be used for adsorption, when using aluminum adjuvant, it is expected to use 5 ⁇ 200 ⁇ g VZV gE can produce high-titer neutralizing antibodies.
- the preferred dose may be 10-100 ⁇ g, and the appropriate immunization dose may be about 10 ⁇ g, 25 ⁇ g, 50 ⁇ g, 100 ⁇ g VZV gE, or about 200 ⁇ g VZV gE, the best dose for adults It may be 50 ⁇ g or 100 ⁇ g VZV gE; the best dose in infants and young children may be 10 ⁇ g, 20 ⁇ g VZV gE.
- the administration mode of the vaccine preparation of the present invention includes, for example, topical, intranasal, mucosal, intradermal, intraperitoneal, subcutaneous and intramuscular injection administration.
- the vaccine formulation of the present invention is optionally combined with adjuvants and/or (other) suitable carriers.
- Suitable protocols for stimulating boosts include intervals of 1, 2, 3, or 6 months between immunizations.
- VZV gE The sequence of the extraenvelope region of VZV gE is a part of the sequence listed in Annex 1 of the present invention.
- the complete sequence of VZV gE was first published in Virus Research (Virus Research, Haumont et al. Vol. 40, 1996p 199-204). Unless the context clearly indicates otherwise, the VZV gE or gE referred to in the following includes truncated VZV gE or other fragments or derivatives of VZV gE-Fc.
- gE or its derivatives or fragments are liquid or lyophilized.
- gE or its derivatives gE-Fc or its fragments, polymers may be present in the suspension containing aluminum adjuvant, or may be present In a solution or suspension containing other immune enhancer components (for example, a solution or suspension containing QS21, cholesterol, mineral oil, vegetable oil, fish oil, long-chain fatty acid, long-chain fatty acid ester, and 3D-MPL adjuvant, etc.).
- gE or its derivatives can be encapsulated in polylactic acid microcarriers, or in microcarriers formed by glycolide/lactide copolymer.
- the formed microcarriers can be slowly injected into the muscle or subcutaneously for a certain period of time. Release the packaged recombinant protein drugs to stimulate the body's immune system to produce antibodies.
- the present invention has the following advantages:
- the present invention uses mammalian animal cells (CHO) to efficiently express the secreted gE-Fc fusion protein, and the obtained target product is glycosylated protein, the expression product is mainly dimer, followed by monomer; each;
- the dimer molecule contains 4 molecules of gE, and the fusion protein produced by the invention has a large molecular weight and strong immunogenicity.
- the gE-Fc of the present invention is a fusion protein between the extra-membrane region of VZV gE and the Fc fragment of human immunoglobulin (CH2-CH3 region).
- Commercially available Protein A affinity chromatography fillers can be used for high-efficiency preliminary separation and purification. , To minimize the emission of environmental pollutants, it is an environmentally friendly separation process, which is conducive to further purification of the target protein by ion exchange chromatography, molecular size exclusion chromatography, etc. in the subsequent steps;
- the recombinant gE-Fc fusion protein expressed by the CHO cell of the present invention is a glycosylated protein, which maintains the spatial structure of the natural gE protein, has good immunogenicity, and has the prospect and advantages of large-scale promotion.
- the Fc of the gE-Fc fusion protein provided by the present invention can bind to the Fc receptors on the surface of antigen-presenting cells existing in the human immune system to actively present gE antigens.
- the results of animal immunogenicity experiments show that using the gE-Fc protein produced by the recombinant CHO cells as the antigen, rabbits and mice can still produce high titer after immunizing rabbits and mice without using oily adjuvants and immune stimulants. Serum neutralizing antibodies;
- Lane 1 DL10000 DNA Marker (10000bp, 7000bp, 4000bp, 2000bp, 1000bp, 500bp and 250bp)
- Lane 2-3 The digested product of plasmid pUC57-gE-Fc
- Lane 1 DL10000 DNA Marker (10000bp, 7000bp, 4000bp, 2000bp, 1000bp, 500bp and 250bp)
- Lane 2-7 Colony PCR screening of 6 randomly picked clones gE-Fc-1 ⁇ 6
- Lane 1 DL10000 DNA Marker (10000bp, 7000bp, 4000bp, 2000bp, 1000bp, 500bp and 250bp)
- Lane 2 Plasmid pXC4-VZV gE-Fc
- Lane 3 Plasmid VZV gE-Fc-straight after linearization
- Figure 8 The geometric mean titers of neutralizing antibodies after immunization of BALB/C mice with different doses of VZV gE aluminum adjuvant vaccine
- the varicella-zoster virus E protein (VZV gE) of the present invention is the extracellular region (ECD, 31-546aa) of the gE protein, with a total of 516 amino acids; the Fc segment is human IgG1Fc, with a total of 232 amino acids (Appendix: Amino acid sequence 1). Connect the varicella-zoster virus E protein gene and the human IgG1 Fc gene in series (Appendix: DNA sequence 2), and entrust Nanjing GenScript Biotechnology Co., Ltd.
- the glycerol strains containing recombinant genes provided by GenScript Biotechnology Co., Ltd. were inoculated into LB (Amp+) medium, cultured at 37°C and 180 rpm for 15 hours, and the plasmid pUC57-gE- was extracted using TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0 Fc;
- the plasmid pUC57-gE-Fc was digested with HindIII and EcoR to obtain the target gene fragment gE-Fc-H/E (approximately 2300bp), and the mammalian cell expression plasmid pXC-K383L was digested to obtain the vector fragment pXC- H/E (size is about 7000bp); the agarose gel electrophoresis detection of the digested product is shown in Figure 1.
- the recovered digested products gE-Fc-H/E and pXC-H/E are ligated by TaKaRa DNA Ligation Kit LONG (TaKaRa) at 16°C for 6 hours and then transformed into competent DH5 ⁇ , 37°C Inverted culture for 15h; two clones gE-Fc-1 and gE-Fc-2 were screened by colony PCR ( Figure 2); PCR amplification was performed with pXC-F and pXC-R as primers and plasmid gE-Fc-1 as template Increase the target gene, the size is about 2400bp; the amplified product was sequenced by Beijing Huada Gene, and the sequence was tested through two additional reactions; the sequencing result was analyzed by the software BioEdit7.0.9.0, and the sequence of gE-Fc-1 was
- the density of viable cells will increase to 5 ⁇ 10 6 ⁇ 7 ⁇ 10 6 cells/ml, add 200ml ⁇ 300ml CD Efficient Feed C, add it once every other day, and measure the glucose content in the culture medium every day. If the glucose content is lower than 11.1mmole/L, add 40% glucose solution to the automatic bioreactor through a peristaltic pump until the glucose content Reach 22mmole/L; culture for 12 to 14 days, terminate the culture when the proportion of live cells drops to 60% to 70%, centrifuge at 12000r/min (or use a 3M depth filter) to remove cells and cell debris, and collect on the cell culture clear.
- the protein solution purified by Protein A affinity chromatography was detected by Shimadzu LC-20AT HPLC (BioCore SEC-500, 7.8mm ⁇ 30cm, Suzhou Nawei Biotechnology Co., Ltd.), and the main peak (dimer) accounted for about 65 % ⁇ 78%, the monomer accounts for about 20% ⁇ 30%. There are still some polymer products before the main peak, which generally accounts for less than 10%.
- the chromatogram is shown in Figure 4.
- Example 8 Lyophilized recombinant VZV gE fusion protein
- the vaccine stock solution obtained in Example 6 was diluted with 20mM Tris-HCl (pH7.2 ⁇ 7.5, containing 135mM NaCl) to 40 ⁇ g/ml ⁇ 800 ⁇ g/ml, and 10% sucrose (or 10% trehalose, 10% mannitol, 10 Lactose) solution to a final concentration of 3%, adjust the concentration of VZV gE in the solution to be dispensed to 20 ⁇ g/ml (or 50, 80, 100, 200, 400 ⁇ g/ml), mix well and distribute into 2ml control bottles , Each bottle of 1.0ml, half-filled with butyl rubber stopper, put it into the freeze-drying bin, set the pre-freezing temperature to -40 ⁇ -45°C, freeze for 4 hours, then start vacuuming and freeze-drying, using automatic heating program control Temperature, increase from -40 ⁇ -25°C for 6 hours, -25 ⁇ -5°C for 4 hours, 0 ⁇ 5°C for 1 hour, 25°C for 1 hour, 35°C for 6
- FIG. 6 shows the serum antibody titers of BALB/C mice immunized with VZV gE aluminum adjuvant vaccine at various doses.
- Serum neutralizing antibody titer determination VZV virus is a virus that can produce cell fusion lesions on human embryonic lung diploid cells. Therefore, the virus plaque reduction neutralization test can be used to test the ability of antibodies with different serum dilutions to neutralize the virus Calculate the serum titer that reduces the number of viruses by 50%.
- the serum antibody neutralization virus test is the most direct test to detect whether there are antibodies that can neutralize the VZV virus in the immunized serum. This test has cumbersome operations, low sensitivity, labor-intensive, time-consuming, and inability to use equipment. Shortcomings such as automatic interpretation, and the calculation of ED 50 requires professional data processing software. The above inconveniences have caused very few people to use this experiment for the determination of serum neutralizing antibodies. Due to the large amount of serum used in this experiment, we only performed neutralizing antibody determinations on the mouse serum collected after the last immunization.
- the positive control serum was rabbit anti-VZV gE serum, and the negative control serum was mixed serum of mice immunized with aluminum adjuvant (1: 10 dilution), each 96-well plate is equipped with 6-well virus solution control wells and 6-well cell control; mix the diluted serum to be tested with a certain amount of OKA strain virus (purchased from ATCC in the United States), and cover the 96-well micro Cover the orifice plate, shake on the microplate shaker for 30 seconds, and then place it in a 10% CO 2 , 37° C. incubator (10%) to react for 30 minutes.
- OKA strain virus purchased from ATCC in the United States
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Abstract
Description
Claims (10)
- 一种重组水痘带状疱疹病毒疫苗制剂,其包括减毒活VZV毒株(OKA株)基因的重组糖蛋白gE胞外区的氨基酸序列和人免疫球蛋白Fc段形成的融合蛋白,所述融合蛋白,其氨基酸序列为SEQ ID No.1。
- 根据权利要求1所述的疫苗制剂,还含有疫苗佐剂。
- 根据权利要求2所述的疫苗制剂,所述疫苗佐剂选自:氢氧化铝佐剂、磷酸铝佐剂,或氢氧化铝和磷酸铝佐剂的混合物。
- 根据权利要求1所述的疫苗制剂,其中每个剂量单位中含有融合蛋白5~200μg。
- 根据权利要求4所述的疫苗制剂,其中每个剂量单位中含有融合蛋白10~100μg。
- 根据权利要求5所述的疫苗制剂,其中每个剂量单位中含有融合蛋白20~60μg。
- 根据权利要求1所述的疫苗制剂,还含有其它能增强免疫原性的物质,包含但不限于:磷脂酰胆碱、卵磷脂、3D-MPL、长链脂肪酸(酯)、矿物油、植物油、甲基纤维素钠、羧甲基纤维素钠、以及含有胆固醇的脂质体的组合物。
- 根据权利要求1所述的疫苗制剂,为冻干制剂。
- 根据权利要求8所述的疫苗制剂,所述冻干制剂,使用前用加入氢氧化铝佐剂混悬液溶解,混合均匀后,肌肉或皮下注射。
- 一种重组基因,其可以表达权利要求1中所述的融合蛋白,其DNA序列为SEQ ID No.2。
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CN116350770B (zh) * | 2021-12-28 | 2024-07-12 | 成都迈科康生物科技有限公司 | 一种带状疱疹疫苗制剂及其制备方法 |
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CN114958882B (zh) * | 2022-05-11 | 2023-05-26 | 晟明生物技术(郑州)有限公司 | 一种表达水痘-带状疱疹病毒gE蛋白的DNA分子 |
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CN116655748A (zh) * | 2023-02-28 | 2023-08-29 | 易慧生物技术(上海)有限公司 | 一种截短型水痘-带状疱疹病毒gE蛋白及其应用 |
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CN116747298B (zh) * | 2023-08-09 | 2024-01-02 | 成都新诺明生物科技有限公司 | 一种水痘-带状疱疹病毒疫苗及其制备方法和应用 |
CN118085111A (zh) * | 2024-04-28 | 2024-05-28 | 天津中逸安健生物科技有限公司 | 一种融合蛋白gE-Fc及其在制备重组带状疱疹疫苗中的应用 |
CN118146319A (zh) * | 2024-05-13 | 2024-06-07 | 普大生物科技(泰州)有限公司 | 一种vzv重组蛋白及其制备方法和应用 |
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