多浪羊IL-8基因原核表达载体的构建与表达

Construction and expression of a prokaryotic expression vector of the IL-8 gene in Duolang sheep

为了成功构建多浪羊白细胞介素-8(IL-8)基因的表达载体并实现其表达,试验根据已发表的绵羊基因序列,在其保守区设计引物,采用RT-PCR方法从多浪羊的外周血淋巴细胞中扩增得到IL-8蛋白基因,此基因的大小为718 bp,经序列比对确定为多浪羊IL-8基因的全编码序列,通过DNA重组技术,将克隆后的IL-8基因质粒转入大肠杆菌,以SalⅠ和XhoⅠ酶切鉴定重组子,以IPTG诱导融合蛋白的表达。结果表明:经SDS-PAGE电泳检测发现,已将IL-8基因定向克隆至原核表达载体BL21(DE3)中,其产物的大小与预期大小相一致,为32.0 ku。说明已成功构建并表达多浪羊IL-8的菌株,并获得了纯化重组多浪羊蛋白。

To successfully construct prokaryotic expression vector of interleukin - 8 （ IL - 8 ） gene in Duolang sheep, the primers were designed in its conserved region, based on the published gene sequences of sheep IL - 8. The protein gene which code the gene of IL - 8 was amplifieded from from peripheral blood of Duolang sheep, by using the technique of RT - PCR, and the size of this gene was 718 bp. The sequence was i- dentified to be the entire coding sequence of IL - 8 gene by using sequence alignment, and the IL - 8 gene plasmid was transferred into E. coli by using technology of the DNA recombinant with enzyme Sal[ and Xho I restriction digestion, and then the fusion protein was induced by IPTG. The results showed that it was found that the gene of IL - 8 was s directionally cloned into prokmyotic expression vector DE3 through the test of SDS - PAGE. The size of the product was 32.0 ku, which was the same with the expected molecular size. It indicates that the IL - 8 is successfully constructed and expressed in Duolang sheep, and the purified recombinant protein of Duolang sheep is obtained.