鸡热休克蛋白70基因的克隆及原核表达

Clone and prokaryotic expression of Hsp70 gene of the broiler

热休克蛋白70基因(heat shock protein 70 gene,Hsp70 gene)具有多种重要生理学功能,为进一步研究鸡Hsp70及其表达蛋白的生物学特性,设计一对特异性引物,并采用SMART技术富集鸡心肌细胞mRNA,RT-PCR扩增Hsp70基因的完整编码区,构建Hsp70原核表达载体,转化E.coli.BL21(DE3),IPTG诱导表达Hsp70重组蛋白.结果显示,扩增片段为1905bp,包含有鸡Hsp70基因完整编码区与GenBank中鸡Hsp70基因的同源性为99.2%～99.8%,SDS-PAGE电泳显示,在92Kda处的蛋白表达量明显增多,Western blot试验证实表达产物能与抗Hsp70阳性血清发生反应,证实本研究扩增出了鸡Hsp70基因的完整编码区,原核表达得到了鸡Hsp70重组蛋白,为进一步研究鸡Hsp70的分子特征及其表达蛋白的生物学功能奠定了基础.

Heat shock protein 70 gene（Hsp70 gene） has a variety of important physiological functions.The purpose of the research is to study the biological characteristics of heat shock protein 70 of the broiler for further.A pair of specific primers are designed and the mRNAs are amplified by SMART？ PCR cDNA Synthesis Kit.Hsp70 gene is amplified by RT-PCR and cloned into expressed vector pET32a（＋）.The recombinant vector pET32a-Hsp70 is constructed and transformed into E.coli BL21（DE3） cells after the induction of isopropyl-B-D-thiogalactoside（IPTG）.The expressed product is analyzed by SDS—PAGE and Western blot.The DNA sequencing result reveals the sequence of amplified Hsp70 gene is 1905bp in length including the complete coding region nucleotide sequence.The gene is highly conserved and shared 99.2 %～99.8 % nucleotide sequence identities with the Hsp70 genes of the broiler strains in GenBank respectively.The SDS—PAGE analysis shows that the fusion protein has the molecular weight（Mv） of a 92 KDa and could react positively with Hsp70 antiserum in Western blot.The study provides a necessary condition for studying the structure,function and clinical application of Hsp70 of the broiler.