含Arg9的人抗HBsAg单链抗体/ EGFP融合蛋白基因的构建、表达和内化作用的研究

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含Arg9的人抗HBsAg单链抗体/ EGFP融合蛋白基因的构建、表达和内化作用的研究

薛茜温伟红孟艳玲薛采芳张勇张巍王涛贾林涛杨安钢

第四军医大学基础部免疫教研室第四军医大学基础部免疫教研室第四军医大学免疫学教研室第四军医大学免疫学教研室第四军医大学免疫学教研室第四军医大学免疫学教研室第四军医大学基础部生物化学和分子生物学教研室第四军医大学基础部生物化学和分子生物学教研室

Construction, expression and Internalization study of human anti-HBsAg single chain antibody/EGFP fusion proteins containing Arg9

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摘要： 目的：构建、表达和纯化带有转膜结构域Arg9的ScFv14/EGFP融合蛋白，并对纯化产物的亲和活性和内化作用进行研究。方法：将Arg9的编码序列分别重组到ScFv14/EGFP基因的5'端或3'端或二者之间，将它们分别克隆入原核表达载体pET32a，转化大肠杆菌BL21(DE3)LysS进行诱导表达和纯化，用间接ELISA方法检测表达产物与HBsAg的亲和活性，并用间接免疫荧光检测纯化蛋白的内化活性。结果：经测序及酶切鉴定证实四种融合基因序列完全正确.SDS-PAGE和Western blot证实四种融合基因成功表达和纯化.间接ELISA检测证实四种融合蛋白均具有HBsAg结合活性，间接免疫荧光检测显示N端带有Arg9的融合蛋白有较强的内化作用，而且不内化进入HBsAg非表达的细胞。结论：成功构建、表达和纯化了ScFv14/EGFP融合蛋白和三种带有Arg9的ScFv14/EGFP融合蛋白，纯化产物均具有与抗原HBsAg亲和的活性，N端带有Arg9的融合蛋白与靶细胞作用有较强的内化作用。

Abstract: To construct，express and purify ScFv14/EGFP fusion proteins which containing Arg9,and to study their binding activities and internalization function. METHODS： Arg9 gene were recombined into 5'terminal、3'terminal of ScFv/EGFP gene or between them before they were cloned into the expression vector pET32a. After induced in E.coli.BL21 and purified, the binding activity and internalization of them were respectively analyzed heir binding activities and internalization function by indirect ELISA and indirect immunofluorescence analysis. RESULTS： DNA sequencing and Restriction endonuclease digestion proved that the four fusion genes were correctly constructed. SDS-PAGE analysis and Western blot showed that they were successfully expressed and purified. Indirect ELISA confirmed that the expressed products had antigen specific binding activity. Indirect immunofluorescence analysis revealed fusion protein which containing Arg9 at its N terminal had much better internalization function，but never internalized into the cells which do not express HBsAg. CONCLUSION：The four fusion genes constructed, expressed and purified successfully. And the purified fusion proteins maintained the binding activity to HBsAg and fusion protein which containing Arg9 at its N terminal had internalization effect much better.

收稿日期: 2006-01-18 出版日期: 2006-07-25

通讯作者: 薛茜

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	温伟红
	薛采芳
	张巍
	王涛
	张勇
	孟艳玲
	薛茜
	贾林涛
	杨安钢

引用本文:

薛茜,温伟红,孟艳玲,薛采芳,张勇,张巍,王涛,贾林涛,杨安钢. 含Arg9的人抗HBsAg单链抗体/ EGFP融合蛋白基因的构建、表达和内化作用的研究[J]. 中国生物工程杂志, .

. Construction, expression and Internalization study of human anti-HBsAg single chain antibody/EGFP fusion proteins containing Arg9. China Biotechnology, .

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https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2006/V26/I07/1

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