新疆卡拉库尔羊TNF-α原核表达及多克隆抗体的制备  

Prokaryotic Expression and Preparation of Polyclonal Antibody TNF-α from Karakul Sheep

作　　者：潘伊微[1] 贺艳艳[1] 潘辉[1] 贾山岭[1] 李莲瑞[2] 

机构地区：[1]塔里木大学生命科学学院,新疆阿拉尔843300 [2]塔里木大学动物科学学院/新疆兵团塔里木畜牧科技重点实验室新疆阿拉尔843300

出　　处：《新疆农业科学》2013年第3期572-577,582,共7页Xinjiang Agricultural Sciences

基　　金：新疆兵团应用基础项目“新疆卡拉库尔羊白细胞功能基因的筛选”(07GC07)

摘　　要：【目的】克隆卡拉库尔羊TNF-α基因,在大肠杆菌中表达、纯化,并对TNF-α蛋白的抗原性进行分析。【方法】采用PCR技术扩增TNF-α基因核酸片段,并克隆入pET-28b表达质粒,在大肠杆菌BL21(DE3)株中表达。用电洗脱法纯化目的蛋白,并用免疫印迹法分析纯化蛋白的抗原特异性。【结果】重组质粒在BL21(DE3)菌株中经IPTG诱导表达一个相对分子质量约为49 KD的融合重组蛋白,原核表达质粒pET-28b-TNF-α表达的目的融合蛋白是以可溶的形式存在。用纯化的pET-28b-TNF-α免疫家兔,在免疫后的第45 d,Western-blotting和琼脂糖扩散试验分析表明,表达的pET-28b-TNF-α能被家兔的阳性血清所识别。【结论】表达蛋白pET-28b-TNF-α具有较好的反应原性和免疫原性。[Objective]The project aims to clone and express the TNF- α protein of B. melitensis in E. coli from Karakul sheep, and meanwhile to purify the expressed protein and detect its immunogenicity. [ Method ] Coding sequences of TNF - α by PCR were cloned in prokaryotic expression vector pET - 28b. Recombinant plasmids were transformed into E. coli BL 21 （ DE3 ） and induced for protein expression by IPTG.. Recombinant proteins were purified by Electroelution and their antigenic specificity was identified by Western blot. [ Result] SDS - PAGE proved that recombinant proteins showed a band with molecular relative mass of 49KD. The prokaryotic expression plasmid pET- 28b -TNF- α existed in the form of the soluble protein. Using the purified TNF - α to immunize rabbits, 45 days after immunization, Western - blotting and the agar diffusion test analysis demonstrated that the expressed TNF -α protein could be detected by positive serum from rabbits and that the expressed pET- 28b- TNF- α protein has good reactivity and immunogenicity.

关 键 词：卡拉库尔羊TNF-α 表达纯化 抗原性分析 

分 类 号：S826[农业科学—畜牧学] S852.16[农业科学—畜牧兽医]