摘要
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 。
Objective To construct a recombinant prokaryotic expression vector (plasmid) containing microneme protein 1 (MIC1) partial gene in toxoplasma gondii (T. gondii) ZS2 isolate. The gene was expressed in varied Escherichia coli (E. coli) after sequencing Methods The gene fragment coding MIC 1 from the genomic DNA of T. gondii ZS2 isolate was amplified by ploymerase chain reaction (PCR). The gene was inserted to a prokaryotic expression vector pWR450 1 by digesting with restriction enzymes and linking reaction. The positive clone was screened on LB plates containing ampicillin and identified by restrictive enzyme digestion,PCR amplification and sequence analysis. The recombinant plasmid was transferred into E.coli TG1,JM109 (DE3) and DH5α,and was expressed under the induction of IPTG. The expression products were identified by SDS PAGE. The MIC1 gene structure was analyzed and compared in homology with the gene sequence of RH isolate using computer software Results The recombinant plasmid pWR450 1/MIC1,after cloning from acquired 471 bp MIC1 gene fragment and amplified from the genome gene ZS2,was complete homologous to the sequence of RH isolate,reflecting its highly conservative. The gene could be expressed as fusion protein with 70 000 in varied E. coli Conclusion Recombinant plasmid pWR450 MIC1 was successfully constructed and could be expressed in different strains of E. coli ,laying a foundation for research on its structure and function.
出处
《中华预防医学杂志》
CAS
CSCD
北大核心
2003年第1期29-32,共4页
Chinese Journal of Preventive Medicine
基金
湖南省教育厅资助 ( 0 0 1C188)
