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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。
Objective To amplify a new gene of microsomal glutathione transferase (mGST) from Clonorchis sinensis, and to determine whether an intron exists and to perform prokaryotic expression and purification. Methods The mGST gene was amplified from the cDNA library and genomic DNA of adult Clonorchis sinensis by PCR. The gene was cloned into the prokaryotic expression vector pET-30a (+) and expressed in Escherichia coli BL21 / DE3, recombinant proteins were purified according to the Ni-NTA agarose instructions and analyzed by SDS-PAGE and TLC. Results The mGST gene was 486 bp in length without any introns. The constructed prokaryotic expression plasmid pET-30a (+) - mGST was expressed in Escherichia coli. The molecular mass unit of the fusion protein was about 24 ku. The recombinant protein reached 11% of the total bacterial protein, and the purity of the purified recombinant protein was 83%. Conclusion The mGST gene has no intron and can be expressed efficiently in Escherichia coli. The purified recombinant protein was obtained, which laid the foundation for further study of its function.