融合蛋白ScFv(3G11)-mms13在大肠杆菌中的表达及鉴定  

The Expression of Fusion Protein ScFv(3G11)-mms13 in E.coli and Its Identification

作　　者：邢秋翠[1] 孔登[1] 纪国强[1] 杨柳青[1] 王小柯[1] 崔楠 

机构地区：[1]潍坊医学院生物化学与分子生物学教研室 [2]95905部队场站卫生队

出　　处：《潍坊医学院学报》2013年第6期407-410,共4页Acta Academiae Medicinae Weifang

基　　金：山东省科技攻关资助课题(课题编号:2009GG10002079)

摘　　要：目的构建抗Ⅳ型胶原酶单链抗体基因ScFv与磁小体膜蛋白基因mms13融合基因的原核表达载体pET30a(+)-ScFv-mms13,在大肠杆菌中诱导表达并对融合蛋白进行初步鉴定。方法 PCR扩增融合基因ScFv-mms13,经EcoRⅠ和XhoⅠ双酶切后克隆入原核表达载pET30a(+)质粒,构建重组表达质粒。将重组质粒导入大肠杆菌DH5a中,利用PCR、酶切筛选鉴定重组菌株。用异丙基疏代半乳糖苷(IPTG)诱导表达融合蛋白,通过改变IPTG浓度、诱导时间优化表达条件。表达产物经SDS-PAGE电泳和Western blot法进行鉴定。结果重组质粒经PCR、酶切、测序证实重组质粒pET30a(+)-ScFv-mms13构建成功。SDS-PAGE电泳结果分析表明诱导菌株在43kDa左右处有明显异源蛋白表达,且确定了在IPTG终浓度为0.2mmol/L、诱导时间为6h时为表达稳定。对表达蛋白进行定位分析确定表达蛋白主要以包涵体形式存在,部分存在于可溶细胞质中。Western blot结果实证该表达产物可与His-tag抗体发生特异性结合,提示为目的融合蛋白。结论成功构建pET30a(+)-ScFvmms13表达载体并表达获得目的融合蛋白。Objective To construct a prokaryotic expression plasmid for ScFv-mms13 fusion gene and identify the expression of fusion protein ScFv-mms13 in E.coli.Methods ScFv-mms13 gene was amplified by PCR and cloned into the prokaryotic expression vector pET30a（＋） to construct recombinant expression plasmid pET 30a（ ＋）-ScFv-mms13.The recombinant plasmid was transformed to E.coli DH5ɑ and identified by PCR and double enzyme digestion .Then the expression vector was transformed to Rosetta （DE3） and the ScFv-mms13 fusion protein was induced expression by IPTG ,optimizing the expression conditions induced by varying the concentration of IPTG ,and induc-tion time.The fusion protein was identified by SDS-PAGE and Western blotting.Results The expressing vector pET30a（ ＋）-ScFv-mms13 was constructed correctly,confirmed by restriction endonuclease digestion and sequencing .The Rosetta（DE3） containing the pET30a（ ＋）-ScFv-mms13 expressed a Mr 43 000 fusion protein under the induction of IPTG .The IPTG final concentration of 0.2mmol/L and inducted time 6h were the optimal expression conditions .The fusion protein mainly existed in inclusion body ,part in the soluble cytoplasm.Western blotting proved that the protein had a specific reaction with His-tag antibody.Conclusion The recombinant expression vector pET 30a（＋）-ScFv-mms13 was successfully constructed and expressed fusion protein .

关 键 词：SCFV mms13 蛋白表达 SDS-PAGE WESTERNBLOT 

分 类 号：R318[医药卫生—生物医学工程]