CAJ | 학술논문

目的扩增华支睾吸虫一个微粒体谷胱甘肽转移酶(mGST)新基因,明确是否存在内含子,并进行原核表达与纯化. 方法用PCR方法分别从华支睾吸虫成虫cDNA(质粒)文库和基因组DNA中扩增mGST基因并测序,将编码基因定向克隆到原核表达载体pET-30a(+),在大肠埃希菌BL21/DE3中表达,按照Ni-NTA agarose说明书纯化重组蛋白,用SDS-PAGE和TLC分析. 结果 mGST基因全长486 bp,没有内含子,构建的原核表达质粒pET-30a(+)-mGST在大肠埃希菌中得到了有效表达,融合蛋白的分子质量单位约为24 ku.重组蛋白达细菌总蛋白质的11%,纯化后的重组蛋白纯度为83%. 结论 mGST基因没有内含子,在大肠埃希菌中能有效表达,得到了纯化的重组蛋白,为进一步研究其功能奠定了基础.
목적확증화지고흡충일개미립체곡광감태전이매(mGST)신기인,명학시부존재내함자,병진행원핵표체여순화. 방법용PCR방법분별종화지고흡충성충cDNA(질립)문고화기인조DNA중확증mGST기인병측서,장편마기인정향극륭도원핵표체재체pET-30a(+),재대장애희균BL21/DE3중표체,안조Ni-NTA agarose설명서순화중조단백,용SDS-PAGE화TLC분석. 결과 mGST기인전장486 bp,몰유내함자,구건적원핵표체질립pET-30a(+)-mGST재대장애희균중득도료유효표체,융합단백적분자질량단위약위24 ku.중조단백체세균총단백질적11%,순화후적중조단백순도위83%. 결론 mGST기인몰유내함자,재대장애희균중능유효표체,득도료순화적중조단백,위진일보연구기공능전정료기출.
Objective To amplify a new encoding gene of microsomal glutathione transferase (mGST) from adult Clonorchis sinensis, determine whether it had intron or not, express it in Escherichia coli and purify the recombinant protein. Methods The gene encoding mGST was obtained from the cDNA (plasmid) library and genomic DNA of adult C. sinensis by PCR and sequenced, respectively. The coding gene was cloned into the prokaryotic expression vector pET-30a(+) and expressed in E. coli BL21/DE3. The recombinant protein was purified according to the protocol of Ni-NTA agarose (QIAGEN, Germany). It was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thin-layer chromatography (TLC). Results The full length of the gene encoding mGST was 486 bp, it had no intron. The recombinant plasmid pET-30a(+)-mGST was efficiently expressed in E. coli, its molecular weight was about 24 ku. The recombinant protein reached up to 11% of total bacterial protein. The purity of the purified recombinant protein was 83%. Conclusion The mGST gene of C. sinensis has no intron. Its efficient expression has been achieved in E. coli. The recombinant protein was purified. These can lay a base for its function research.