Arg9/人抗HBsAg单链抗体/ EGFP融合蛋白基因的构建、表达及活性分
作者：薛茜，温伟红，孟艳玲，张勇，张巍，王涛，张瑞，杨安钢【关键词】 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： 构建带有转膜结构域Arg9编码序列的R9/ScFv14/EGFP融合基因，将其转化入大肠杆菌中进行表达，并分析表达产物与HBsAg的'结合活性. 方法： 设计引物，将Arg9的编码序列引入单链抗体基因的5′端，PCR扩增后获得带有Arg9编码序列的ScFv14基因，将PCR产物连入pMD18T载体，进行序列测定. 将测序正确的ScFv14基因和R9/ScFv14基因分别与EGFP基因连接后转化入原核表达载体pET32a，获得ScFv14/EGFP和R9/ScFv14/EGFP两种融合基因的表达载体，转化大肠杆菌BL21(DE3)LysS，以IPTG诱导表达，对表达产物进行SDSPAGE分析，并用间接ELISA方法检测其与HBsAg的亲和活性. 结果： 经酶切鉴定及测序证实ScFv14/EGFP融合基因和R9/ScFv14/EGFP融合基因序列完全正确.SDSPAGE分析表明两种融合基因在大肠杆菌BL21中成功获得表达，表达量分别占菌体总蛋白的20% 和25%.间接ELISA检测证实所表达的ScFv14/EGFP融合蛋白和R9/ScFv14/EGFP融合蛋白均具有HBsAg结合活性. 结论： 成功构建了带有转膜结构域Arg9编码序列的R9/ScFv14/EGFP融合基因，并在大肠杆菌BL21中成功表达，表达产物ScFv14/EGFP和R9/ScFv14/EGFP均具有与抗原HBsAg特异性结合的活性.
【关键词】 肝炎，乙型；肝炎表面抗原，乙型；ScFv；Arg9
0引言
单链抗体（ScFv）是抗体重链可变区(VH)与轻链可变区(VL)通过一段连接肽(linker)连接而成的一种小分子抗体.其分子质量仅为完整抗体的1/6，不仅能较好地保持抗原结合能力，并且具有分子质量小、穿透力强、半衰期短、免疫原性低、制备流程简单等特点，更有利于体内应用［1-3］. 绿色荧光蛋白(green fluorescent protein, GFP) 是从水母中分离出来的一种发光蛋白,可在A450 nm～490 nm的蓝光激发下发出绿色荧光，使用普通荧光显微镜即可进行观察.是近年来细胞生物领域中应用最为广泛的标记性蛋白质之一.EGFP为一种突变型GFP，所产生的荧光比野生型GFP增强，可作为
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