HPV31 L1 C端截短基因可在昆虫细胞表达体系中高水平制备L1 VLP疫苗
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摘要
目的利用昆虫细胞表达体系制备人乳头瘤病毒(HPV)31 L1病毒样颗粒(VLP)疫苗。方法化学合成法获得昆虫Sf9细胞偏性密码子优化的、C端删除27个氨基酸编码序列的HPV31 L1截短基因(HPV31 L1MΔC),构建重组杆状病毒,感染Sf9细胞进行表达;分别采用超声破碎、含离子型表面活性剂(1%SDS等)或含非离子型表面活性剂(0.5%TritonX-100)的裂解缓冲液,制备细胞裂解上清;氯化铯超速离心法纯化后经动态光散射及透射电子显微镜鉴定;0.1μg的HPV31 L1MΔC VLP于第0和2周肌肉免疫BALB/c小鼠,第4周采集血清分析中和活性。结果 3种裂解方法制备的上清均显示HPV31 L1MΔC蛋白的特异表达,其表达量约占裂解上清总蛋白的10%;采用非离子型表面活性剂获得的裂解上清纯化后,目的蛋白纯度高,产量达11.5 mg/L;纯化蛋白的水化分子直径为103.3 nm,均一性好;电镜分析为直径50～60 nm的VLP。免疫血清中HPV31中和抗体滴度为1 440。结论 HPV31 L1MΔC在昆虫细胞表达体系制备的VLP产量高、免疫原性好,可用于生产含HPV31 L1 VLP的多价疫苗。
Objective To produce human papillomavirus(HPV) type 31 L1 virus-like particle(VLP) vaccine in baculovirus system. Methods A C-terminal 27 residues truncated HPV31 L1 gene(HPV31 L1 MΔC) which optimized with Sf9 cell bias condons, was synthesized and expressed in baculovirus system. Cells infected with HPV31 L1 MΔC recombinant baculovirus were lysed by sonification, or incubation with either ionic surfactant-containing buffer or nonionic surfactant-containing buffer, and the supernatants were harvested respectively for expression assays. The supernatants prepared by incubation with nonionic surfactant-containing buffer were purified by CsCl ultracentrifugation and HPV31 L1 MΔC protein was identified by dynamic light scatter(DLS) and transmission electronic microscopy(TEM). BALB/c mice were immunized with 0.1 μg HPV31 L1 MΔC VLP intramuscularly at weeks 0, 2 and sera at week 4 were taken for neutralization assay against HPV31 pseudovirus. Results HPV31 L1 MΔC protein was expressed in all three kinds of supernatants prepared by either sonification or incubation with two different surfactant-containing buffer,and amounted for 10% of the total proteins. The yield of purified HPV31 L1 MΔC was up to 11. 5 mg per liter suspension medium. HPV31 L1 MΔC protein showed an average hydrodynamic diameter of 103. 3 nm with a mean polydispersity index of 0. 190 by DLS,and presented VLP with an average diameter of 50 ～ 60 nm which is similar to native virions shown by TEM. HPV31 L1 MΔC VLP induced high titer of HPV31 neutralizing antibody( 1 440) in mice. Conclusions The VLP produced with HPV31 L1 MΔC truncated gene in baculovirus system is a promising candidate for development of multivalent HPV L1 VLP vaccine for its high yield and potent immunogenicity.
Objective To produce human papillomavirus(HPV) type 31 L1 virus-like particle(VLP) vaccine in baculovirus system. Methods A C-terminal 27 residues truncated HPV31 L1 gene(HPV31 L1 MΔC) which optimized with Sf9 cell bias condons, was synthesized and expressed in baculovirus system. Cells infected with HPV31 L1 MΔC recombinant baculovirus were lysed by sonification, or incubation with either ionic surfactant-containing buffer or nonionic surfactant-containing buffer, and the supernatants were harvested respectively for expression assays. The supernatants prepared by incubation with nonionic surfactant-containing buffer were purified by CsCl ultracentrifugation and HPV31 L1 MΔC protein was identified by dynamic light scatter(DLS) and transmission electronic microscopy(TEM). BALB/c mice were immunized with 0.1 μg HPV31 L1 MΔC VLP intramuscularly at weeks 0, 2 and sera at week 4 were taken for neutralization assay against HPV31 pseudovirus. Results HPV31 L1 MΔC protein was expressed in all three kinds of supernatants prepared by either sonification or incubation with two different surfactant-containing buffer,and amounted for 10% of the total proteins. The yield of purified HPV31 L1 MΔC was up to 11. 5 mg per liter suspension medium. HPV31 L1 MΔC protein showed an average hydrodynamic diameter of 103. 3 nm with a mean polydispersity index of 0. 190 by DLS,and presented VLP with an average diameter of 50 ～ 60 nm which is similar to native virions shown by TEM. HPV31 L1 MΔC VLP induced high titer of HPV31 neutralizing antibody( 1 440) in mice. Conclusions The VLP produced with HPV31 L1 MΔC truncated gene in baculovirus system is a promising candidate for development of multivalent HPV L1 VLP vaccine for its high yield and potent immunogenicity.
引文
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[5] Einstein MH, Levin MJ, Chatterjee A, et al. Comparative humoral and cellular immunogenicity and safety of human papillomavirus (HPV)-16/18 AS04-adjuvantedvaccine and HPV-6/11/16/18 vaccine in healthy women aged 18-45 years:Follow-up through Mouth 48 in a Phase III randomized study[J]. Hum Vaccines Immunother, 2014, 10:3455-3465.
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[7] 高波,张靖,张学锋,等.人乳头瘤病毒31和33型L1蛋白类病毒颗粒的制备及其免疫原性[J]. 中国生物制品学杂志, 2014, 27:1508-1516.
[8] Chen X, Liu H, Wang Z, et al. Human papillomavirus 16L1-58L2 chimeric virus-like particles elicit durable neutralizing antibody responses against abroad-spectrum of human papillomavirus types[J]. Oncotarget, 2017, 8:63333-63344.
[9] Zhou J, Doorbar J, Sun XY, et al. Identification of the nuclear localization signal of human papillomavirus stype 16 L1 protein[J]. Virology,1991, 185:625-632.
[10] Varsani A, Williamson AL, Jaffer MA, et al. A deletion and point mutation study of the human papillomavirus type 16 major capsid gene[J]. Virus Res,2006, 122:154-163.
[11] Paintsil J, Muller M, Picken M, et al. Carboxyl terminus of bovine papillomavirus type-1 L1 protein is not required for capsid formation[J]. Virology, 1996,223:238-244.
[12] Zhang T, Xu Y, Qiao L, et al. Trivalent Human Papillomavirus (HPV) VLP vaccine covering HPV type 58 can elicit high level of humoral immunity but also induce immune interference among component types[J]. Vaccine, 2010,28:3479-3487.
[2] Bruni L, Barrionuevo-Rosas L, Albero G, et al. Human papillomavirus and related Disease report-world[J]. ICO HPV Information Centre, 2016, 1:1-60.
[3] Buck CB, Day PM, Trus BL, et al. The papillomavirus major capsid protein L1.[J] Virology, 2013, 445:169-174.
[4] Schiller JT, Castellsague X, Garland SM, et al. A review of clinical trials of human papillomavirus prophylactic vaccines[J]. Vaccine,2012, 30:123-138.
[5] Einstein MH, Levin MJ, Chatterjee A, et al. Comparative humoral and cellular immunogenicity and safety of human papillomavirus (HPV)-16/18 AS04-adjuvantedvaccine and HPV-6/11/16/18 vaccine in healthy women aged 18-45 years:Follow-up through Mouth 48 in a Phase III randomized study[J]. Hum Vaccines Immunother, 2014, 10:3455-3465.
[6] Wang D, Fan F, Li Z, et al. Stop codon mutagenesis for homogeneous expression of human papillomavirus L1 protein in Escherichia coli[J]. Protein Expr Purif, 2017, 133:110-120.
[7] 高波,张靖,张学锋,等.人乳头瘤病毒31和33型L1蛋白类病毒颗粒的制备及其免疫原性[J]. 中国生物制品学杂志, 2014, 27:1508-1516.
[8] Chen X, Liu H, Wang Z, et al. Human papillomavirus 16L1-58L2 chimeric virus-like particles elicit durable neutralizing antibody responses against abroad-spectrum of human papillomavirus types[J]. Oncotarget, 2017, 8:63333-63344.
[9] Zhou J, Doorbar J, Sun XY, et al. Identification of the nuclear localization signal of human papillomavirus stype 16 L1 protein[J]. Virology,1991, 185:625-632.
[10] Varsani A, Williamson AL, Jaffer MA, et al. A deletion and point mutation study of the human papillomavirus type 16 major capsid gene[J]. Virus Res,2006, 122:154-163.
[11] Paintsil J, Muller M, Picken M, et al. Carboxyl terminus of bovine papillomavirus type-1 L1 protein is not required for capsid formation[J]. Virology, 1996,223:238-244.
[12] Zhang T, Xu Y, Qiao L, et al. Trivalent Human Papillomavirus (HPV) VLP vaccine covering HPV type 58 can elicit high level of humoral immunity but also induce immune interference among component types[J]. Vaccine, 2010,28:3479-3487.
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