重组海参溶菌酶基因工程菌的构建及原核表达

Construction of recombinant bacteria for sea cucumber lysozyme and its prokaryotie expression

根据GenBank中海刺参溶菌酶的cDNA序列(Accession no.EF036468),利用RT-PCR技术扩增出海刺参溶菌酶的基因片段(SL),将其克隆到原核表达载体pET-32a(+),得到重组质粒pET-32a(+)-SL,再转化至E.coil Rosetta(DE3)pLysS。利用IPTG诱导表达,重组蛋白经SDS-PAGE分析,得到一条分子质量约为31ku的特异条带。经过Western blotting分析鉴定,结果显示重组海参溶菌酶成功在大肠杆菌中表达,并且有部分可溶性蛋白,占总菌体蛋白约10%。这些结果为进一步研究海参溶菌酶的作用机理奠定基础。

The cDNA of Stichopus japonicus lysozyme(SL) was cloned by the RT-PCR technique according to accession no.EF036468published in GenBank.The DNA fragment of SL was subclone into the expression vector of pET-32a(+) to construct the recombinant plasmid of pET32a(+)-SL. Then the recombinant plasmids were transformed into Escherichia coli Rosetta(DE3) pLysS.After IPTG induction, the recombination protein is an specific strap that the molecular weight is about 31ku by the analysis of SDS-PAGE.The recombinant SL could be successfully expressed and partially existed in soluble form, the soluble SL of which was about 10% of the total protein.These results will provide a basis to further study of sea cucumber lysozyme mechanism.