本文關(guān)鍵詞：呼吸道合胞病毒融合蛋白人源性單鏈抗體的構(gòu)建及初步鑒定　出處：《安徽醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人呼吸道合胞病毒 F蛋白 噬菌體抗體庫(kù) 單鏈抗體

【摘要】： 目的:人呼吸道合胞病毒(Human Respiratory Syncytial Virus,RSV)廣泛分布于世界各地,是導(dǎo)致嬰幼兒嚴(yán)重下呼吸道感染最重要的病毒病原。病毒感染機(jī)體后的免疫保護(hù)機(jī)制尚未明確,也無(wú)特異性防治方法。在RSV所編碼的11種蛋白中,融合糖蛋白(fusion glycoprotein,F)和粘附糖蛋白(attachment glycoprotein,G)是僅有的兩種中和抗原,而F蛋白在RSV不同亞型間具有高度的抗原同源性,是主要的交叉保護(hù)性抗原。本研究嘗試?yán)檬删w展示技術(shù)構(gòu)建大容量天然人源性噬菌體抗體庫(kù),從中篩選出RSV F蛋白的人源性單鏈抗體(single chain Fv fragment, scFv),為RSV感染的預(yù)防及特異性治療等研究奠定基礎(chǔ)。 方法:分離10名健康供者外周血淋巴細(xì)胞,從中提取細(xì)胞總RNA,通過RT-PCR擴(kuò)增人抗體VH、VL基因,并利用SOE-PCR將其拼接組裝成scFv基因。將scFv基因克隆入pCANTAB5E噬菌粒載體,通過電轉(zhuǎn)化E.coli TG1感受態(tài)細(xì)胞,獲得大容量噬菌體抗體庫(kù),再經(jīng)輔助噬菌體M13K07挽救重組噬菌粒。利用生物淘洗方法,使抗體庫(kù)中特異性識(shí)別RSV F蛋白的噬菌體scFv得到富集,并利用噬菌體ELISA方法篩選出陽(yáng)性克隆,陽(yáng)性克隆進(jìn)行核酸序列分析。將陽(yáng)性克隆噬菌體感染E.coli HB2151,經(jīng)IPTG誘導(dǎo),制備RSV F蛋白的可溶性單鏈抗體,并以Western及Dot blot方法進(jìn)行初步分析鑒定。 結(jié)果:RT- PCR法擴(kuò)增出全套人抗體VH(約350 bp)、VL(約320 bp)片段,利用SOE-PCR方法在體外成功拼接成scFv基因(約750 bp)并克隆入pCANTAB5E噬菌粒載體,構(gòu)建了庫(kù)容為1.8×107的大容量人源性天然噬菌體scFv庫(kù)。經(jīng)過五輪生物淘洗,特異性識(shí)別RSV F的scFv得到了108倍的富集,噬菌體ELISA篩選出18株陽(yáng)性克隆,取OD值最高的克隆E4經(jīng)測(cè)序并檢索Kabat數(shù)據(jù)庫(kù)分析,顯示其基因與人免疫球蛋白可變區(qū)基因具有高度同源性,Western及Dot blot分析表明單鏈抗體已成功表達(dá)。 結(jié)論:構(gòu)建了大容量天然人源噬菌體scFv庫(kù),從中成功篩選出能特異性識(shí)別RSV F的scFv,并進(jìn)行了可溶性表達(dá)和初步鑒定,為進(jìn)一步的生物學(xué)活性研究奠定了基礎(chǔ)。
[Abstract]:Objective: human Respiratory Syncytial virus (RSVV) is widely distributed all over the world. It is the most important virus pathogen leading to severe lower respiratory tract infection in infants. The mechanism of immune protection after virus infection is not clear, and there is no specific prevention and cure method. Among the 11 proteins encoded by RSV. Fusion glycoprotein F) and adhesion glycoprotein attachment (glycoprotein). G) is the only two neutralizing antigens, and F protein has high antigenic homology among different subtypes of RSV. This study attempts to construct a large capacity natural human phage antibody library using phage display technology. Single chain Fv fragment (scFv) of RSV F protein was screened. To lay a foundation for the prevention and specific treatment of RSV infection. Methods: peripheral blood lymphocytes from 10 healthy donors were isolated and total RNAs were extracted from them. The VHV L gene of human antibody was amplified by RT-PCR. The scFv gene was cloned into pCANTAB5E phagocyte vector and transformed into E. coli TG1 receptive cells. A large phage antibody library was obtained, and then the recombinant bacteriophage was saved by auxiliary phage M13K07. The phage scFv, which specifically recognizes RSV F protein, was enriched in the antibody library, and the positive clones were screened by phage ELISA method. The positive clones were infected with E. coli HB2151 and the soluble scFv of RSV F protein was prepared by IPTG induction. The method of Western and Dot blot was used to analyze and identify. Results A complete set of human antibody VH (about 350bp) was amplified by using the method of: RT- PCR. The scFv gene (about 750bp) was successfully spliced into pCANTAB5E vector by SOE-PCR in vitro. A large human natural phage phage scFv library with a capacity of 1.8 脳 10 ~ 7 was constructed. After five rounds of biological washing, the specific RSV F recognition scFv was 108-fold enriched. Eighteen positive clones were screened by phage ELISA. The clone E4 with the highest OD value was sequenced and analyzed by Kabat database. Western and Dot blot analysis showed that its gene was highly homologous to the variable region gene of human immunoglobulin, and the single chain antibody was successfully expressed. Conclusion: a large capacity natural human phage scFv library was constructed, from which scFv, which could specifically recognize RSV F, was successfully screened, and the soluble expression and preliminary identification were carried out. It lays a foundation for the further study of biological activity.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

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