小鼠Lewis肺癌细胞与旋毛虫相关抗原基因sHSPs的原核表达及鉴定

Prokaryotic expression and identification of sHSP genes for antigens associated with Lewis lung cancer (LLC) cells and Trichinella spiralis

目的对小鼠Lewis肺癌(LLC)细胞与旋毛虫(Trichinella spiralis)相关抗原基因sHSPs进行原核表达,对表达产物进行抗原性鉴定。方法利用噬菌体文库展示技术筛选旋毛虫与LLC细胞相关抗原基因并分析,获得sHSPs(DQ 986457)基因。以旋毛虫cDNA为模板,PCR扩增sHSPs基因,连接至表达载体pET-32a(+)后转化入感受态BL21(DE3),以IPTG诱导表达,采用SDS-PAGE和Western blot鉴定重组蛋白的表达及其反应原性。结果重组表达质粒pET-32a(+)-sHSPs经双酶切及测序鉴定证明克隆载体构建正确,SDS-PAGE检测其表达重组蛋白相对分子质量约为36×10~3。Western blot检测重组蛋白可被抗LLC细胞多抗血清识别。结论成功构建了pET-32a(+)-sHSPs克隆载体,其表达的重组蛋白可与抗LLC细胞多抗血清反应即具有反应原性,为该蛋白的研究奠定了基础。

Objective To express and identify sHSP genes for antigens associated with Trichinella spiralis and Lewis lung cancer(LLC)cells.Method Phage display technology was used to screen for genes for antigens associated with Trichinella spiralis and LLC cells.The sequence of an identified gene was aligned with the sequence published in GenBank,and the gene was analyzed.An sHSP(DQ 986457)gene was obtained from a T.spiralis cDNA expression library.The sHSP gene was amplified with PCR using T.spiralis cDNA as a template and ligated into the expression vector pET-32 a(+).The recombinant vector pET-32 a(+)-sHSP was transformed into competent BL21(DE3)cells,and expression of the protein was induced with IPTG.Expression of the recombinant protein and its reactogenicity were identified using SDS-PAGE and Western blotting.Result The expression vector was verified to be correctly constructed after the recombinant plasmid pET-32 a(+)-sHSP was identified using BamH I and Sal I digestion and sequencing.The molecular weight of the recombinant sHSP of the expressed product was about 36 x10^3.The recombinant sHSP was detected by anti-LLC cell antiserum according to Western blotting.Conclusion The expression vector pET-32 a(+)-sHSP was successfully constructed.The expressed recombinant sHSP was recognized by anti-LLC cell antiserum,which proves that recombinant sHSP is reactive.This finding lays the foundation for the study of this protein.