真核细胞表达单纯疱疹病毒I型包膜糖蛋白B及其抗原性与免疫原性分析

中国生物工程杂志

2010, Vol. 30

Issue (06): 8-13

研究报告

真核细胞表达单纯疱疹病毒I型包膜糖蛋白B及其抗原性与免疫原性分析

王正茂¹,李琳²,管文燕²,李越希^1,2**

1.南京军区军事医学研究所南京 210002
2.南京医科大学基础医学院生物化学与分子生物学系南京 210029

Expression of Herpes Simplex Virus Type I Glycoprotein B in Eukaryotic Cells and Analysis of Its Antigenicity and Immunogenicity

WANG Zheng-mao¹,LI Lin²,GUAN Wen-yan²,LI Yue-xi^1,2

1.East-China Institute for Medical Biotechniques, Nanjing 210002,China
2.Department of Biochemistry & Molecular Biology,School of Preclinical Medicine, Nanjing Medical University, Nanjing 210029, China

全文: PDF(852 KB) HTML

摘要：

为在真核细胞中表达并纯化I型单纯疱疹病毒（HSV I）包膜糖蛋白gB，并分析其抗原性和免疫原性，化学合成了包膜糖蛋白gB1胞外区基因片段，构建真核表达载体，并转染至HEK293细胞，表达的蛋白用羊抗HSV1+HSV2血清作为一抗，用ELISA检测其抗原性；用纯化的gB1蛋白免疫昆明小鼠，观察诱发抗体产生的时间及其效价，并用ELISA和Western blot检测小鼠抗gB1多克隆抗体特异性识别重组gB1抗原的能力，评价其免疫原性。结果显示在HEK293细胞中成功表达重组gB1蛋白，ELISA证实羊抗HSV1+HSV2多抗能够识别重组gB1蛋白；重组gB1蛋白免疫小鼠7周后，小鼠血清中多克隆抗体效价达到5×103，表明在真核细胞中高效表达并纯化的重组gB1蛋白具有良好的抗原性和免疫原性，为HSV检测试剂和疫苗研究提供了理论基础。

关键词： 单纯疱疹病毒; 包膜糖蛋白gB1; 真核表达; 抗原性; 免疫原性

Abstract:

To express and purify herpes simplex virus type I (HSV I) glycoprotein B (gB) in eukaryote cells with the purpose of analyzing the antigenicity and immunogenicity. At the beginning, the extracellular domain fragment gene of gD1 was synthesized by chemical method and cloned into eukaryotic expression vector pCEP4 to construct the recombinant plasmid pCEP4-gD1. After transfection of HEK293 cells with the recombinant plasmid, the expressed protein was characterized by Western blot and purified through Ni affinity chromatography. Then antigenicity of the protein was detected by ELISA. Finally, the purified protein was used to immunize Kunming mice in 1, 3, 5 weeks respectively, and antiserum were collected in 3, 5 and 7 weeks. The antibody titer was detected by indirect ELISA for immunogenicity analysis. Gene sequencing analysis demonstrates that the recombinant plasmid pCEP4-gB1 was constructed successfully. Western blot analysis indicated one major protein band, which molecular weight is approximate 85 kDa corresponding to the truncated forms of gB1 protein, was observed. In addition, ELISA detection showed that expressed gB1 has good antigenicity. After the third immunization, antibody titer of the mouse anti-gB1 was 5×103. The successful expression of the recombinant protein gD1, which can induce humoral immune response, lays a foundation for serological diagnosis and vaccine study of HSV.

Key words: Herpes simplex virus Type I (HSV I) Glycoprotein B Eukaryotic cell expression Antigenicity Immunogenicity

收稿日期: 2010-01-11 出版日期: 2010-06-12

基金资助:

国家“863”计划资助项目(2006AA02A226)

通讯作者: 李越希 E-mail: liyxi2007@126.com

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	王正茂
	李琳
	管文燕
	李越希

引用本文:

王正茂李琳管文燕李越希. 真核细胞表达单纯疱疹病毒I型包膜糖蛋白B及其抗原性与免疫原性分析[J]. 中国生物工程杂志, 2010, 30(06): 8-13.

WANG Zheng-Mao, LI Lin, GUAN Wen-Yan, LI Huo-Xi. Expression of Herpes Simplex Virus Type I Glycoprotein B in Eukaryotic Cells and Analysis of Its Antigenicity and Immunogenicity. China Biotechnology, 2010, 30(06): 8-13.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I06/8

［1］ Eizuru Y. Development of new antivirals for herpesviruses. Antivir Chem Chemother, 2003, 14(6):299308.
［2］ Wald A, Link K. Risk of human immunodeficiency virus infection in herpes simplex virus type 2seropositive persons: a metaanalysis. J Infect Dis, 2002, 185(1):4552.
［3］ Perez G, Skurnick J H, Denny T N, et al. Herpes simplex type II and Mycoplasma genitalium as risk factors for heterosexual HIV transmission: report from the heterosexual HIV transmission study. Int J Infect Dis, 1998, 3(1):511.
［4］ Chen C Y, Ballard R C, BeckSague C M, et al. Human immunodeficiency virus infection and genital ulcer disease in South Africa: the herpetic connection. Sex Transm Dis, 2000, 27(1):2129.
［5］ Langenberg A G M, Burke R L, Adair S F, et al. A recombinant glycoprotein vaccine for herpes simplex type2: safety and efficacy. Ann Intern Med, 1995, 122(12):889898.
［6］杨慧兰，周翠，关蕾，等．单纯疱疹病毒II型CTL表位DNA疫苗的Th1/Th2免疫应答研究中国皮肤性病学杂志,2007,21(7):402404 Yang H L, Zhou C, Guan L, et al. Chin J Derm Venereol, 2007, 21(7):402404.
［7］吕敏，潘明洁，李越希，等．单纯疱疹病毒1型糖蛋白B胞外区基因片段的克隆、表达及初步鉴定．药物生物技术，2008，15(2):9093. Lv M, Pan M J, Li Y X, et al. Pharmaceutical Biotechnology, 2008, 15(2):9093.
［8］ Ashley R L, Wald A. Genital herpes: review of the epidemic and potential use of typespecific serology. Clin Microbiol Rev, 1999, 12(1):18.
［9］ Stanberry L, Cunningham A, Mertz G, et al. New developments in the epidemiology, natural history, and management of genital herpes. Antiviral Res, 1999, 42(1):114.
［10］ Fleming D T, McQuillan G M, Johnson R E, et al. Herpes simplex virus type 2 in the United States, 1976 to 1994. N Engl J Med, 1997, 337(16):11051111.
［11］ Ribes J A, Steele A D, Seabolt J P, et al. Sixyear study of the incidence of herpes in genital and nongenital cultures in a central Kentucky medical center patient population. J Clin Microbiol, 2001, 39(9):33213325.
［12］ Tayal S C, Pattman R S. High prevalence of herpes simplex virus type 1 in female anogenital herpes simplex in NewcastleuponTyne 198392. Int J STD AIDS, 1994, 5(5):359361.
［13］ Thompson C. Genital herpes simplex typing in genitourinary medicine: 1995–1999. Int J STD AIDS, 2000, 11(8):501502.
［14］ Dingwell K S, Brunetti C R, Hendricks R L, et al. Herpes simplex virus glycoproteins E and I facilitate celltocell spread in vivo and across junctions of cultured cells. J Virol, 1994, 68(2):834845.
［15］ Spear P G. Membrane fusion induced by herpes simplex virus. In: Bentz J, editor. Viral Fusion Mechanisms. CRC Press, Boca Raton, 1993. 201232.
［16］ Berman P W, Dowbenko D, Lasky L(A) Detection of antibodies to herpes simplex virus with a continuous cell line expressing cloned glycoprotein(D) Science, 1983, 222(4623):524527.
［17］ Glorioso J, Schroder C H, Kumel G, et al. Immunogenicity of herpes simplex virus glycoproteins gC and gB and their role in protective immunity. J Virol, 1984, 50(3):805812.
［18］ Little S P, Jofre J T, Courtney R J, et al. A virion associated glycoprotein essential for infectivity of herpes simplex virus type 1. Virology, 1981, 115(1):149160.
［19］ Watson R J, Weis J H, Salstrom J S, et al. Herpes simplex virus type 1 glycoprotein D gene: nucleotide sequence and expression in Escherichia coli. Science, 1982, 218(4570):381384.
［20］ Pellett P E, Kousoulas K G, Pereira L, et al. Anatomy of the herpes simplex virus 1 strain F glycoprotein B gene: primary sequence and predicted protein structure of the wild type and of monoclonal antibodyresistant mutants. J Virol, 1985, 53(1):243253.
［21］ Nass P H, Elkins K L, Weir J P. Protective immunity against herpes simplex virus generated by DNA vaccination compared to natural infection. Vaccine, 2001, 19 (1112):15381546.
［22］ Chen Y, Hu D, Eling D J, et al. DNA vaccines encoding fulllength or truncated Neu induce protective immunity against Neuexpressing mammary tumors. Cancer Res, 1998, 58(9):19651971.
［23］ Lin X H, Ali M A, Openshaw H, et al. Deletion of the carboxyterminus of herpes simplex virus type 1 (HSV1) glycoprotein B does not affect oligomerization, heparinbinding activity, or its ability to protect against HSV challenge. Arch Virol, 1996, 141(6):11531165.

[1]	程旭,杨雨睛,吴赛男,侯勤龙,李咏梅,韩慧明. 金黄色葡萄球菌SarA、IcaA及其融合基因的DNA疫苗构建及在小鼠免疫应答中的初步研究 ^*[J]. 中国生物工程杂志, 2020, 40(7): 41-50.
[2]	岑黔鸿,高彤,任怡,雷涵. 重组酿酒酵母表达幽门螺杆菌VacA蛋白及其免疫原性分析^*[J]. 中国生物工程杂志, 2020, 40(5): 15-21.
[3]	齐家龙, 高瑞雨, 靳输梅, 高福兰, 杨旭, 马雁冰, 刘存宝. 水痘-带状疱疹病毒糖蛋白E在昆虫细胞中的表达、鉴定及免疫原性分析 ^*[J]. 中国生物工程杂志, 2019, 39(8): 17-24.
[4]	杜力,刘晓志,高健,王志明. 抗体药物免疫原性评估的研究进展[J]. 中国生物工程杂志, 2018, 38(2): 89-94.
[5]	郎巧利,余琳,何麒麟,葛良鹏,杨希. 高效构建卵清白蛋白scFv噬菌体文库及其筛选 ^*[J]. 中国生物工程杂志, 2018, 38(11): 25-31.
[6]	吴剑荣,彭星桥,詹晓北. 聚唾液酸,一种非GAGs、非免疫原性生物材料的应用研究进展 ^*[J]. 中国生物工程杂志, 2017, 37(12): 96-102.
[7]	王晶, 夏文跃, 潘小霞, 赵冰心, 滕玉梅, 李传印, 杨筱舟, 文喻玲, 陈元鼎. 研究报告Ⅱ型脊灰病毒抗原表位嵌合蛋白的免疫学研究[J]. 中国生物工程杂志, 2016, 36(8): 1-8.
[8]	夏文跃, 王晶, 赵冰心, 潘小霞, 文喻玲, 陈元鼎. 轮状病毒VP4抗原表位在VP6载体蛋白同一位点表达比较研究[J]. 中国生物工程杂志, 2015, 35(8): 9-15.
[9]	王青, 徐彦召, 魏晓晓, 王秋霞, 杭柏林, 孙亚伟, 王飞飞, 胡建和. 猪繁殖与呼吸综合征病毒GP5a多克隆抗血清的制备[J]. 中国生物工程杂志, 2015, 35(8): 38-43.
[10]	李多, 杨丽娟, 赵玉娇, 潘玥, 陈俊英, 付娟娟, 黄新伟, 邱丽娟, 孙强明. 研究报告重组Ⅱ型登革病毒NS1的表达及其免疫原性的研究[J]. 中国生物工程杂志, 2014, 34(9): 4-8.
[11]	王金胜, 姜浩武, 张晋霞, 潘磊, 赵凤芝, 于云飞, 蔡亚雄, 邓宁. 抗FGF2人鼠嵌合抗体在HEK293T细胞中的表达优化[J]. 中国生物工程杂志, 2014, 34(5): 14-22.
[12]	毛红艳, 马正海. 单纯疱疹病毒1型囊膜糖蛋白gD在大肠杆菌中的表达、纯化及其免疫活性的鉴定[J]. 中国生物工程杂志, 2014, 34(11): 54-59.
[13]	庞敏, 王海龙, 郭民, 郭睿. 人ANKRD49基因真核表达载体的构建及其功能的初步研究和RNA干扰靶点的鉴定[J]. 中国生物工程杂志, 2014, 34(10): 15-21.
[14]	钟菲菲, 杨慧兰, 吕芳彪, 薛礼长, 白利利, 樊建勇. 单纯疱疹病毒2型潜伏相关转录本开放读码框的表达及其抗凋亡功能分析[J]. 中国生物工程杂志, 2013, 33(10): 36-43.
[15]	刘玉芬, 冬黎黎, 孙玉刚, 刘鹏, 陈辉, 赵文阁. 中介蝮蛇毒纤溶酶基因真核表达载体的构建及其功能的研究[J]. 中国生物工程杂志, 2012, 32(10): 1-6.

Viewed

Full text

Abstract

Cited

Shared

Discussed