猪热休克蛋白70基因的克隆及在大肠杆菌中的表达

Cloning and expression of porcine heat shock protein 70 gene in Escherichia coli

根据GenBank中收录的猪HSP70基因序列,设计了1对特异性引物。运用RT-PCR技术从经热应激诱导的大约克猪组织总RNA中扩增得到了HSP70基因,并将其克隆至pMD18-T Simple载体上,进行序列测定。将编码猪HSP70的基因亚克隆至pET-32a(+)上,并将重组质粒pET-32-pHSP70在大肠杆菌BL21(DE3)pLysS中经IPTG诱导表达。核苷酸测序结果显示,获得的基因序列全长2200 bp,含1932bp的开放阅读框,编码643个氨基酸,与已知猪HSP70核苷酸序列和氨基酸序列的同源性都高达99.5%。SDS-PAGE电泳及Western-blot分析表明,猪HSP70在BL21(DE3)pLysS中得到了高效表达。

Based on the sequences of porcine heat shock protein 70（pHSP70） gene published in Gen- Bank,a pair of specific primers was designed and synthesized. The cDNA of pHSP70 was amplified by RT- PCR from the total RNA extracted from the tissue which was obtained from Yorkshire sow with heat stress,and then the pHSPT0 cDNA was cloned into the pMD18-T Simple vector and sequenced, Sequencing analysis showed that the acquired entire cDNA was 2 200 bp including the ORF of pHSP70, which encoded 643 amino acid residues,and it was proved to be sequence of pHSP70 because of 99.5% homology with the published pHSP70 nucleotide sequence. Then ORF of pHSP70 was subcloned into pET-32a（-） vector,the recombinant plasmid pET 32-pHSP70 was constructed and then transformed into BL21 （DE3） pLysS competent cell and induced with IPTG. SDS-PAGE and Western blotting confirmed that the pHSP70 was expressed in pET-32-pHSP70 transformed BL21（DE3）pLysS.