本文關鍵詞：鼠源抗2型單純皰疹病毒gB蛋白噬菌體單鏈抗體庫的構建及特異性抗體篩選　出處：《浙江工業(yè)大學》2011年碩士論文　論文類型：學位論文

  更多相關文章： 單純皰疹病毒(HSV) gB蛋白 噬菌體抗體庫 單鏈抗體(ScFv)

【摘要】：單純皰疹病毒2型(HSV-2)是線性雙鏈DNA病毒,是引起生殖器皰疹(GH)的主要病原體。HSV-2感染患者后,病毒移行至感覺神經節(jié)中并在背側根神經節(jié)中形成潛伏感染。孕婦感染HSV-2后,常常導致意外流產以及新生兒皰疹。現(xiàn)已證明,HSV-2與HIV的感染有關系,HSV-2將加重HIV感染后的癥狀。 包膜糖蛋白是HSV感染發(fā)病的關鍵所在,在HSV-2編碼的糖蛋白中gB、gD蛋白是病毒囊膜的主要成分,在病毒復制和刺激中和抗體的產生中起重要作用,是HSV主要的免疫原,能誘導體液和細胞免疫,產生高滴度中和抗體,激發(fā)DTH反應及T細胞增殖反應。 目的：構建鼠源性抗Ⅱ型單純皰疹病毒(HSV-2) gB蛋白噬菌體單鏈抗體庫,并從中篩選特異性的抗體。方法：應用重組噬菌體抗體技術,用Ⅱ型單純皰疹病毒的包膜糖蛋白B的氨基端抗原決定簇融合蛋白(Trx-gBN)免疫BALB/c小鼠,取脾分離淋巴細胞,提取總RNA, RT-PCR分別擴增抗體重鏈可變區(qū)基因(VH)、輕鏈可變區(qū)基因(VL),SOE-PCR法拼接成ScFv基因,將其克隆入噬粒載體pCANTAB-5E中,電轉化于E.coli TG1,通過輔助噬菌體M13K07援救得到噬菌體單鏈抗體庫。并以Ⅱ型單純皰疹病毒的包膜糖蛋白B的氨基端抗原決定簇融合蛋白(Trx-gBN)為篩選靶位,淘選陽性重組噬菌體,經鑒定后對其進行序列分析,非競爭ELISA,初步檢測重組ScFv的特異性抗原結合活性和中和活性等。結果：成功地構建了庫容量約為4×109的噬菌體ScFv庫,菌落PCR分析得重組率為93%。經過三輪的篩選,我們篩選到了具有病毒中和活性的抗HSV-2特異性ScFv。結論：成功的構建噬菌體展示ScFv庫,獲得了鼠源抗HSV-2特異性ScFv,為將來進一步研究抗HSV2-gB的治療性人源化抗體奠定了基礎。
[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.
【學位授予單位】：浙江工業(yè)大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392

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