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Arg9/人抗HBsAg單鏈抗體/ EGFP融合蛋白基因的構建、表達及活性分
作者：薛茜，溫偉紅，孟艷玲，張勇，張巍，王濤，張瑞，楊安鋼【關鍵詞】 肝炎,乙型；肝炎表面抗原,乙型；ScFv；Arg9
【Abstract】 AIM: To construct R9/ScFv14/EGFP fusion genes and to analyze the binding activity of the fusion proteins after they were expressed in E.coli BL21. METHODS: A series of oligonucleotide primers were designed and used to amplify the genes of ScFv14 and R9/ScFv14. The PCR products were cloned into pMD18T vector, followed by DNA sequencing. R9/ScFv14 gene and ScFv14 gene were ligated with EGFP gene respectively before they were recombined into the expression vector pET32a. After induced in E.coli BL21 by IPTG, the expressed fusion proteins named R9/ScFv14/EGFP and ScFv14/EGFP were detected by SDSPAGE and the binding activity of them were analyzed by indirect ELISA. RESULTS: Restriction endonuclease digestion and DNA sequencing proved that the two fusion genes were correctly constructed. SDSPAGE analysis showed that they were successfully expressed in E.coli BL21 and the percentages of them were 20% and 25% of total bacteria proteins respectively. Indirect ELISA confirmed that the expressed products had antigen specific binding activity. CONCLUSION: The two fusion genes were constructed successfully. And the products of them expressed in E.coli BL21 maintained the binding activity to HBsAg.
【Keywords】 hepatitis B; hepatitis B surface antigens; ScFv; Arg9
【摘要】 目的： 構建帶有轉膜結構域Arg9編碼序列的R9/ScFv14/EGFP融合基因，將其轉化入大腸桿菌中進行表達，并分析表達產物與HBsAg的結合活性. 方法： 設計引物，將Arg9的編碼序列引入單鏈抗體基因的5′端，PCR擴增后獲得帶有Arg9編碼序列的ScFv14基因，將PCR產物連入pMD18T載體，進行序列測定. 將測序正確的ScFv14基因和R9/ScFv14基因分別與EGFP基因連接后轉化入原核表達載體pET32a，獲得ScFv14/EGFP和R9/ScFv14/EGFP兩種融合基因的表達載體，轉化大腸桿菌BL21(DE3)LysS，以IPTG誘導表達，對表達產物進行SDSPAGE分析，并用間接ELISA方法檢測其與HBsAg的親和活性. 結果： 經酶切鑒定及測序證實ScFv14/EGFP融合基因和R9/ScFv14/EGFP融合基因序列完全正確.SDSPAGE分析表明兩種融合基因在大腸桿菌BL21中成功獲得表達，表達量分別占菌體總蛋白的20% 和25%.間接ELISA檢測證實所表達的ScFv14/EGFP融合蛋白和R9/ScFv14/EGFP融合蛋白均具有HBsAg結合活性. 結論： 成功構建了帶有轉膜結構域Arg9編碼序列的R9/ScFv14/EGFP融合基因，并在大腸桿菌BL21中成功表達，表達產物ScFv14/EGFP和R9/ScFv14/EGFP均具有與抗原HBsAg特異性結合的活性.
【關鍵詞】 肝炎，乙型；肝炎表面抗原，乙型；ScFv；Arg9
0引言
單鏈抗體（ScFv）是抗體重鏈可變區(qū)(VH)與輕鏈可變區(qū)(VL)通過一段連接肽(linker)連接而成的一種小分子抗體.其分子質量僅為完整抗體的1/6，不僅能較好地保持抗原結合能力，并且具有分子質量小、穿透力強、半衰期短、免疫原性低、制備流程簡單等特點，更有利于體內應用［1-3］. 綠色熒光蛋白(green fluorescent protein, GFP) 是從水母中分離出來的一種發(fā)光蛋白,可在A450 nm～490 nm的藍光激發(fā)下發(fā)出綠色熒光，使用普通熒光顯微鏡即可進行觀察.是近年來細胞生物領域中應用最為廣泛的標記性蛋白質之一.EGFP為一種突變型GFP，所產生的熒光比野生型GFP增強，可作為
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