蚓激酶基因在大肠杆菌中的表达

目的]实现蚓激酶基因在大肠杆菌中的高效表达。方法]采用RT-PCR技术,以含蚓激酶基因的质粒pMD18-Tf3为模板进行RT-PCR扩增,克隆了蚓激酶基因Tfc,并进行测序,之后将蚓激酶基因Tfc克隆到大肠杆菌表达载体pBV220,转化大肠杆菌DH5α,用LB培养基于30℃培养24 h后,然后调温至42℃继续培养2 h诱导蚓激酶基因表达,收集菌体,进行SDS-PAGE电泳,利用纤维平板法检验蚓激酶的活性。结果]测序发现该基因全长729 bp,共编码243个氨基酸,GenBank登录号为EU167735。SDS-PAGE电泳表明诱导表达后菌体总蛋白在28 kD处有一特异性条带,而含空质粒pBV220的大肠杆菌经诱导表达后无该条带,表达蛋白主要以包涵体形式存在,无纤维蛋白酶活性。结论]该研究为创新蚓激酶的生产工艺提供了依据。

Expression of Lumbrokinase Gene in Escherichia coli

Objective] The study was to implement high level expression of lumbrokinase gene in Escherichia Coli.Method] With RT-PCR technique,the plasmid pMD18Tf3 which contained lumbrokinase gene was taken as the template to do RT-PCR amplification and the lumbrokinase gene Tfc was cloned.And then the lumbrokinase gene Tfc was cloned into E.coli expression vector pBV220 to transform E.coli DH5α.The E.coli DH5α was first cultured on LB medium at 30 ℃ for 24 h,and then continuously cultured at 42 ℃ for 2 h to induce the express of lumbrokinase gene.The thallus was collected to do SDS-PAGE electrophoresis and the activity of lumbrokinase was detected with fibrin plate method.Result] The gene sequencing showed that the full length of lumbrokinase gene was 729 bp,it totally coded 243 amino acid,and its accession number in GenBank was EU167735.The SDS-PAGE electrophoresis showed that after inducing expression,the total bacterial protein revealed a specific band at 28 kD,but the E.coli which contained empty plasmid had no this band after inducing expression.The expression protein mainly existed as an inclusion body,and it had no fibrinolysin activity.Conclusion] The study provided a basis for the innovation of the production technology for lumbrokinase.