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周期型马来丝虫HSP70基因原核表达载体的构建及表达产物的分析

Expression of periodic Brugia malayi HSP70 gene and expressed protein analysis by SDS-PAGE

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【作者】 方政吴建军陈阳黄为群姜声扬石佑琴

【Author】 FANG Zheng WU Jian-jun CHEN Yang HUANG Wei-qun JIANG Sheng-yang SHI You-qin Department of Parasitology,School of Basic Medical Scielwes,Nantong University,Nantong 226001,China

【机构】 南通大学基础医学院寄生虫学教研室南通大学基础医学院寄生虫学教研室

【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。

【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.

【基金】 江苏省教育厅自然科学研究计划项目(02KJD310002)
  • 【文献出处】 中国地方病学杂志 ,Chinese Journal of Endemiology , 编辑部邮箱 ,2006年05期
  • 【分类号】R392
  • 【被引频次】3
  • 【下载频次】56
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