【摘要】 目的在大肠埃希菌宿主系统中表达周期型马来丝虫热休克蛋白70(HSP70)基因,并对表达产物进行SDS-PAGE分析。方法以重组质粒pGEM-T-HSP70为模板,根据报道的HSP70基因序列设计合成一对引物,用PCR法扩增HSP70基因(包括完整开放读码框架片段及其上下游序列共长2 198 bp),PCR产物定向插入原核表达载体pGEX-4T-3中,构建的重组质粒pGEX-4T-3-HSP70经双酶切鉴定。将重组质粒转化大肠埃希菌BL21(DE3),用IPTG诱导GST．Tag融合蛋白的表达,经SDS-PAGE分析并经凝胶图像分析确定目的蛋白的表达水平。结果重组表达质粒pGEX-4T-3-HSP70全长约7 200 bp,经双酶切后应得到长度为4 968 bp的载体和2 198 bp的目的基因2个片段,1%琼脂糖凝胶电泳显示结果同预期完全相符。HSP70相对分子质量(Mr)为70×10~3,质粒pGEX-4T-3的融合部分(GST．Tag)表达产物的Mr约为26×10~3,将重组质粒转化BL-21 (DE3)菌,经IPTG诱导表达,菌体蛋白的SDS-PAGE图谱显示,诱导组出现特异性蛋白表达条带,其Mr约100×10~3,与预计大小相同。目的蛋白占菌体总蛋白的12%左右。结论成功地构建了重组原核表达载体pGEX-4T- 3-HSP70：周期型马来丝虫HSP70基因可在大肠埃希菌中稳定表达,为制备和纯化表达蛋白以及丝虫病疫苗的进一步研究打下基础。更多还原

【Abstract】 Objective To express periodic Brugia malayi Heat-Shock proteins 70(HSP70)gene,whose GenBank accession number was M68933,in Escherichia coli and to analyze the protein by SDS-PAGE.Methods HSP70 gene open reading frame fragment was obtained by PCR method,template was pGEM-T-HSP70 plasmid and two primers were synthesized according to Brugia malayi HSP70 cDNA.PCR product and pGEX-4T-3 plasmid were digested with two restriction endonuclease SalⅠand NotⅠ.DNA fragment was recovered from agarose gel.The recovery products were ligated by T4 DNA ligase.The recombinant plasmid pGEX-4T-3-HSP70 was transferred into Escherichia coli BL21(DE3)strain and was induced by IPTG to express GST.Tag fusion protein during culturing then analyzed by SDS-PAGE.The level of the expressed protein was determined by Image system.Results The recombinant expression plasmid pGEX-4T-3-HSP70 was digested with two restriction endonuclease Sd I and Not I, two DNA fragments were obtained as expected.HSP70 gene was expressed efficiently in Escherichia coll.SDS- PAGE showed that Escherichia coil transferred with pGEX-4T-3-HSP70 plasmid expressed an extra band at about 100×10~3.The fusion protein accounted for approximately 12% of the total germ protein.Conclusions pGEX-4T- 3-HSP70 plasmid has been constructed successfully and HSP70 gene has been expressed successfully in Escherichia coli,which provides a basis for preparation and purification of proteins and for producing antifilarial vaccine.更多还原