斯氏艾美耳球虫热休克蛋白70基因的克隆及表达分析

Cloning and expression analysis of heat shock protein 70(HSP70) gene from Eimeria stiedai

为了获得斯氏艾美耳球虫(E.stiedai)的热休克蛋白70(HSP70)的全基因序列,并诱导表达重组HSP70蛋白,试验根据免疫蛋白组学研究的质谱结果获得的部分氨基酸序列和已发布近源物种HSP70蛋白序列设计引物,通过c DNA末端快速扩增法(RACE)获得E.stiedai HSP70的全基因序列,将其连入原核表达载体p ET-28a(+),转化Competent Rosetta2(DE3)p Lys S,通过IPTG诱导表达重组HSP70蛋白,并进行Western-blot分析。结果表明:HSP70序列全长为2 727 bp,最大ORF为2 010 bp。HSP70在诱导后37℃培养4 h条件下有表达,但全部为不溶性表达;在诱导后20℃过夜培养条件下无表达。说明试验成功获得E.stiedai HSP70的全基因序列以及重组HSP70蛋白。

To obtain the whole genome sequence of heat shock protein 70 （HSP70） of Eimeria stiedai （ E. stiedai） and induce the expression of recombinant protein HSP70, a pair of special primers was designed based on the partial amino acid sequences obtained from the mass spectrometry results of immunological proteomics and the published HSP70 nucleotide sequence of near - source species of E. stiedai. The whole genome sequence of HSP70 of E. stiedai was obtained by the rapid amplification of cDNA ends （RACE）. HSP70 gene was ligated into prokaryotic expression vector pET -28a（ ＋ ）, and transformed to Competent Rosetta2 （DE3）pLysS. The recombinant protein was expressed by inducing with IPTG and analyzed by Western - blot. The results showed that the full - length sequence of HSP70 was 2 727 bp, and the largest open reading frame （ORF） was 2 010 bp. HSP70 was expressed at 37℃ for 4 h after induction , but total expression was insoluble, and there was no expression cultured at 20 ℃ overnight after induction. The results indicate that the whole gene sequence of HSP70 of E. stiedai and recombinant protein HSP70 are successfully obtained in the test.