猪α干扰素基因的克隆、原核表达及生物学活性鉴定  

Cloning,expression,identification and potency analisis of gene encoding poIFN-α

作　　者：夏俊保[1] 赵俊[1] 汤仁树[1] 崔寰[1] 陈伟[1] 王明丽[1] 

机构地区：[1]安徽医科大学微生物学教研室,合肥230032

出　　处：《安徽医科大学学报》2009年第1期53-56,共4页Acta Universitatis Medicinalis Anhui

基　　金：安徽省教育厅自然科学基金重点项目(编号:KJ2008A085)

摘　　要：目的构建稳定、高效表达的重组猪α干扰素(rpoIFN-α)大肠杆菌表达菌株,并纯化鉴定该菌株表达的目的蛋白,进行生物学活性检测。方法提取猪肝脏细胞基因组DNA,PCR扩增猪α干扰素(poIFN-α)基因,克隆到pMD18-T载体上,测序鉴定后再亚克隆到pGEX-5X-1载体上,转化入E.coliBL21中后得到表达菌株,用IPTG诱导表达,收集的菌体超声破碎后取上清得粗制蛋白,用GST亲和层析法进行纯化,产物用Western blot进行鉴定,并在Hep-2/VSV系统上滴定效价。结果重组质粒pGEX-5X-1/poIFN-α经过EcoRⅠ和XhoⅠ限制性酶切后可见501bp的目的片段,提取质粒测序鉴定,结果与GeneBank中的序列一致。转化入E.coliBL21中32℃条件下经IPTG诱导5h,提取总蛋白,SDS-PAGE检测显示带有GST标记的rpoIFN-α能高效表达,分子量约45ku,可溶性rpoIFN-α约占菌体蛋白的0.35,Western-blot鉴定显示有特异性条带。效价滴定结果表明该rpoIFN-α具有抗病毒活性,纯化后效价为7.5×106IU/ml,比活性达到1.1×107IU/mg。结论成功克隆了poIFN-α基因,并可在大肠杆菌中高效表达具有抗病毒活性的rpoIFN-α。Objective To construct stable, high efficiency expressing gene engineering bacteria of recombinant polFN-α （rpoIFN-α）, purify and identify the rpoIFN-α expressed by it. Methods The gene of poIFN-α was amplified from pig hepatic cell chromosomal by PCR and inserted into the pMD18-T vector, then subcloned into pGEX- 5X-1 vector after identified by sequencing. The recombinant plasmid was transformed into the E. coli BI21, then the rpoIFN was induced, expressed and purified by GST affinity chromatograph, identified by western blot. The potency of rpoIFN-α was determined on the system of Hep-2/VSV. Results The swine interferon α gene was success- fully isolated. Recombinant E. coli BI21 strain expressing rpoIFN-α was obtained, and the dissolved rpoIFN-α was about 0.35 of the tropina. The relative molecular mass of rpol FN-α was 45 000 dalton, and was equal to the expected molecular weight. There was specific strap in western blot analysis. Detected through the Hep-2/VSV system, the potency of rpolFN-α was 7.5 ×10^6 IU/ml, and the specific activity was 1.1 × 10^7 IU/mg. Conclusion The poIFN-α gene is successfully cloned and the recombination E. coli BL21 that expresshigh activity rpoIFN-α efficiently was obtained.

关 键 词：干拢素α/遗传学 重组 遗传 猪 大肠杆菌/遗传学 

分 类 号：R978.7[医药卫生—药品] R394-3[医药卫生—药学]