鸡肺表面活性蛋白A的可溶性表达及活性检测  

Soluble expression and activity detection of chicken pulmonary surfactant protein A

作　　者：黄琪[1,2] 汪凯[1,2] 潘玲[1] 祁克宗[1] 李泽君[2] 陈鸿军[2] 刘红梅[1] 

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国兽医科学》2015年第11期1196-1200,共5页Chinese Veterinary Science

基　　金：安徽省自然科学基金项目(1408085MC49);中央公益性单位专项经费(2015JB15);中国农业科学院动物流感病原生态学创新团队专项经费

摘　　要：利用RT-PCR扩增鸡肺表面活性蛋白A(cSP-A)基因,将其连接pMD19-T Simple载体,经测序正确后,扩增成熟肽基因片段并酶切克隆入pCold-TF原核表达载体中,转化到感受态宿主菌BL21(DE3)中,加入不同浓度异丙基硫代半乳糖苷(IPTG)于16℃诱导表达24h,超声波裂解,通过His·Tag纯化试剂盒纯化,分别进行SDS-PAGE检测。结果扩增得到615bp的成熟肽基因片段;SP-A蛋白获得可溶性表达,融合蛋白的大小为80ku。Western-blot结果显示,该蛋白可被特异性多克隆抗体识别。抑菌试验结果显示,与纯化的天然cSP-A蛋白不同,cSP-A原核表达产物并不具备抑菌活性。Chicken pulmonary surfactant protein A（cSP-A）gene was amplified by RT-PCR and then subcloned into pMD19-T Simple vector.After sequencing,the mature cSP-A fragment without the signal peptide sequence was transferred into the prokaryotic expression vector,pCold-TF.The final construct is designated and cloned into pCold-cSPA.The recombinant plasmid was transformed into Escherichia coli BL21（DE3）competent cells.The bacteria were induced with isopropyl-β-D-1-thiogalactopyranoside at 16℃for 24 hours.The products were sonicated and then purified by His-Tag purification kit.The results showed that the fused protein were soluble expressed with a size of 80 ku by SDS-PAGE analysis and by Western-blot.However,the fused peptide had no activities to inhibit the tested bacteria.

关 键 词：鸡肺表面活性蛋白A 原核表达 可溶性表达 活性检测 

分 类 号：S852.45[农业科学—基础兽医学]