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目的 构建弓形虫ZS2株pWR450 1 微线体蛋白 1 (MIC1 )原核表达重组质粒 ,对MIC1基因进行序列测定 ,并在不同大肠杆菌中表达。方法 用PCR技术从弓形虫ZS2株的基因组DNA中扩增编码MIC1的基因片段 ,酶切 ,连接 ,重组入pWR450 1表达载体 ,再经含氨苄培养基筛选、酶切、PCR鉴定 ,进行序列测序后转化大肠杆菌TG 1、JM1 0 9(DE3)和DH5α。在不同菌体浓度及不同剂量异丙基 β D 硫代半乳糖诱导下 ,用SDS 聚丙烯酰胺凝胶电泳鉴定MIC1融合蛋白的表达。 结果 从ZS2株基因组DNA中扩增出特异的MIC1基因片段 ,克隆后获得pWR450 1 /MIC1重组质粒 ,测序结果表明 ,MIC1这部分基因与弓形虫RH株相应基因序列完全一致 ,高度保守。该基因可在不同大肠杆菌中表达相对分子质量约 70 0 0 0的融合蛋白。结论 构建了弓形虫ZS2株pWR450 1 MIC1重组质粒 ,为研究MIC1的结构与功能奠定了基础
Objective To construct the recombinant plasmids of pWR450 1 microtubule protein 1 (MIC1) of ZS2 strain of Toxoplasma gondii and to determine the expression of MIC1 gene in different E.coli. Methods The gene fragment encoding MIC1 was amplified from the genomic DNA of Toxoplasma gondii ZS2 by PCR. The fragment was digested, ligated and recombined into pWR450 1 expression vector. The recombinant plasmid was screened by ampicillin medium, identified by restriction enzyme digestion and PCR, sequenced Escherichia coli TG1, JM1 0 9 (DE3) and DH5α were then transformed. The expression of MIC1 fusion protein was identified by SDS polyacrylamide gel electrophoresis under different bacterial concentrations and different doses of isopropyl β D thiogalactose. Results The specific fragment of MIC1 was amplified from the genomic DNA of ZS2 strain. The recombinant plasmid pWR450 1 / MIC1 was cloned. The sequence of MIC1 gene was identical with that of RH strain Toxoplasma gondii RH. The gene can be expressed in different E. coli relative molecular mass of about 70 0 0 0 fusion protein. Conclusion The pWR450 1 MIC1 recombinant plasmid of Toxoplasma gondii ZS2 was constructed, which laid the foundation for studying the structure and function of MIC1