鼠源抗2型单纯疱疹病毒gB蛋白噬菌体单链抗体库的构建及特异性抗体筛选

发布时间：2017-12-31 17:42

本文关键词：鼠源抗2型单纯疱疹病毒gB蛋白噬菌体单链抗体库的构建及特异性抗体筛选　出处：《浙江工业大学》2011年硕士论文　论文类型：学位论文

【摘要】：单纯疱疹病毒2型(HSV-2)是线性双链DNA病毒,是引起生殖器疱疹(GH)的主要病原体。HSV-2感染患者后,病毒移行至感觉神经节中并在背侧根神经节中形成潜伏感染。孕妇感染HSV-2后,常常导致意外流产以及新生儿疱疹。现已证明,HSV-2与HIV的感染有关系,HSV-2将加重HIV感染后的症状。包膜糖蛋白是HSV感染发病的关键所在,在HSV-2编码的糖蛋白中gB、gD蛋白是病毒囊膜的主要成分,在病毒复制和刺激中和抗体的产生中起重要作用,是HSV主要的免疫原,能诱导体液和细胞免疫,产生高滴度中和抗体,激发DTH反应及T细胞增殖反应。目的：构建鼠源性抗Ⅱ型单纯疱疹病毒(HSV-2) gB蛋白噬菌体单链抗体库,并从中筛选特异性的抗体。方法：应用重组噬菌体抗体技术,用Ⅱ型单纯疱疹病毒的包膜糖蛋白B的氨基端抗原决定簇融合蛋白(Trx-gBN)免疫BALB/c小鼠,取脾分离淋巴细胞,提取总RNA, RT-PCR分别扩增抗体重链可变区基因(VH)、轻链可变区基因(VL),SOE-PCR法拼接成ScFv基因,将其克隆入噬粒载体pCANTAB-5E中,电转化于E.coli TG1,通过辅助噬菌体M13K07援救得到噬菌体单链抗体库。并以Ⅱ型单纯疱疹病毒的包膜糖蛋白B的氨基端抗原决定簇融合蛋白(Trx-gBN)为筛选靶位,淘选阳性重组噬菌体,经鉴定后对其进行序列分析,非竞争ELISA,初步检测重组ScFv的特异性抗原结合活性和中和活性等。结果：成功地构建了库容量约为4×109的噬菌体ScFv库,菌落PCR分析得重组率为93%。经过三轮的筛选,我们筛选到了具有病毒中和活性的抗HSV-2特异性ScFv。结论：成功的构建噬菌体展示ScFv库,获得了鼠源抗HSV-2特异性ScFv,为将来进一步研究抗HSV2-gB的治疗性人源化抗体奠定了基础。
[Abstract]:Herpes simplex virus type 2 (HSV-2), a linear double-stranded DNA virus, is the main pathogen causing genital herpes herpes. The virus moves to the sensory ganglion and forms a latent infection in the dorsal root ganglion. The infection of HSV-2 in pregnant women often leads to accidental abortion and neonatal herpes. There is a relationship between HSV-2 and HIV infection. HSV-2 may aggravate the symptoms after HIV infection. Envelope glycoprotein is the key to the pathogenesis of HSV infection. In the glycoprotein encoded by HSV-2, gBGD protein is the main component of viral envelope. It plays an important role in virus replication and stimulation of neutralizing antibody production. It is the main immunogen of HSV, which can induce body fluid and cell immunity and produce high titer neutralizing antibody. DTH reaction and T cell proliferation were stimulated. Objective: to construct murine anti-herpes simplex virus (HSV-2) GB single-chain antibody library and screen specific antibodies. Methods: recombinant phage antibody technique was used. BALB/c mice were immunized with the amino terminal antigen determinant of herpes simplex virus envelope glycoprotein B (Trx-gBN). Spleen lymphocytes were isolated and total RNA was extracted. The heavy chain variable region gene of antibody was amplified by RT-PCR, and the light chain variable region gene was spliced into ScFv gene by SOE-PCR. It was cloned into the phagocyte vector pCANTAB-5E and transformed into E. coli TG1. The phage scFv library was obtained by assisting bacteriophage M13K07, and the fusion protein Trx-gBN was used as the amino terminal antigen determinant of herpes simplex virus (HSV) envelope glycoprotein B. To screen the target. Amoy positive recombinant phage was identified and sequenced, non-competitive ELISA. The specific antigen-binding activity and neutralization activity of recombinant ScFv were preliminarily detected. Results: a phage phage ScFv library with a capacity of about 4 脳 10 ~ 9 was successfully constructed. Colony PCR analysis showed that the recombination rate was 93%. We screened out the specific HSV-2 specific scFv.Conclusion: the phage display ScFv library was successfully constructed and mouse anti-#en2# specific ScFv was obtained. It will lay a foundation for the further study of therapeutic humanized antibody against HSV2-gB in the future.
【学位授予单位】：浙江工业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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