奶牛IL-6基因克隆与原核表达

Cloning and Prokaryotic Expression of Gene Coding IL-6 of Holstein Cows

目的:克隆新疆地区奶牛il-6基因,获得原核表达IL-6蛋白。方法:根据GenBank上牛IL-6的序列,用premier5.0软件设计一对引物。以牛肺巨噬细胞提取的RNA反转录产物(cDNA)为模板扩增出567bp的条带,克隆到pBS-T载体,测序正确后,提取质粒经BamHⅠ和EcoRⅠ双酶切回收目的条带,亚克隆到pGEX-4T-1原核表达载体,转化DE3菌,经IPTG诱导表达。以表达产物免疫小白鼠血清为一抗检测表达产物的反应原性。结果:成功克隆了新疆地区奶牛il-6基因,克隆部分编码188个氨基酸与GenBank公布的序列100%同源,表达出46kDa的融合蛋白,免疫印迹检测显示原核表达牛IL-6有免疫原性。结论:新疆地区奶牛在IL-6的基因序列上与其他地方牛无差异,原核表达奶牛IL-6蛋白为在奶牛乳房炎疫苗中的佐剂效果研究打下了基础。

Objective：Cloned and prokaryotic expression of gene coding IL-6 of cows.Method：Sepecies-specific primers were designed by Primer 5.0 sofeware accroding to the il-6 gene sequenses of bovine available in genebank.The 576bp fragments were amplified and obtained by PCR using the first-strand cDNA,which was generated from bovine pulmonary macrophage cell RNA.The PCR products for il-6 of bovine were cloned into pBS-T vector to confirm the suquense,digested with EcoRⅠ and BamHⅠ,Correct clones were then subcloned into the pGEX-4T-1 expression vector and then transformed into DE3 E.coli.Which induced by IPTG,the expression products digested with western blotting.Result：The bovine IL-6 gene sequence got was from Xinjiang cows and a 46kDa fusion protein,Western blot shows that it could be recognized by specific antibodies.Conclusion：There was no different of IL-6 gene sequence between xinjiang Holstein cows and other regions.The prokaryotic expression IL-6 protein provide a basis for the using as a adjuvant in mastits vaccine.