水蛭源性类胰蛋白酶抑制剂表达载体的构建与表达

Cloning and expression of leech derived tryptase inhibitor

目的:构建水蛭源性类胰蛋白酶抑制剂(leech derived tryptase inhibitor,LDTI)原核表达载体,并在大肠埃希菌[BL21(DE3)]中高效表达重组蛋白。方法:以含有目的核酸序列的质粒为模板,通过PCR技术扩增出LDTI蛋白编码序列,构建含目的片段的T克隆载体以及原核表达载体pET28a-LDTI重组质粒亚克隆,进行限制性内切酶双酶切鉴定和核酸序列分析,然后将该质粒转化大肠埃希菌BL21(DE3),IPTG诱导,以Tricine-SDS-PAGE和蛋白质印迹方法分析和鉴定重组蛋白的表达。结果:成功构建了水蛭源性类胰蛋白酶抑制剂原核表达载体pET28a-LDTI,并获得相应的融合蛋白;本实验证明,LDTI在大肠埃希菌工程菌中高效表达,在温度为33℃,IPTG浓度为0.7 mmol,诱导时间为1 h,所表达的目的蛋白约占菌体总蛋白的15%左右。结论:成功构建了LDTI原核表达载体并使其在原核系统中高效表达,为进一步研究水蛭源性类胰蛋白酶抑制剂的生化特性和生物学功能奠定了良好的基础。

Objective： To construct the prokaryotic expression vector of LDTI gene and express LDTI protein in E. coli[ BL21 （DE3） ]. Methods： A 141 bp fragment of the LDTI gene was amplified by PCR technique from the template of the plasmid containing LDTI gene,then it was cloned into T vector.After amplified and digested with restricted enzyme,the target fragment was subcloned into plasmid pET28a,the recombinant plasmid was transferred into E.coli Jm109.The fragment was identified by restricted enzyme and nucleotide sequencing.The recombinant expression plasmid containing LDTI gene was transformed into E.coli BL21（DE3） and the target protein expression was induced by isopropyl-B-D-thiogalactopyranoside （IPTG） and confirmed by Tricine-SDS-PAGE and Western blot.Results：The recombinant plasmid of pET28a-LDTI was obtained by PCR and identified by sequencing.The target protein was expressed abundantly in E.coli BL21（DE3） and the yield of LDTI protein was accounted for approximately 15% of germ proteins after one hour＇s induction by 0.7mmol/L IPTG at 33℃.Conclusion：pET28a-LDTI was successfully constructed and can highly express objective protein in BL21（DE3）,which will further help to the bioactivity of LDTI studying.