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.
出处
《新疆农业科学》
CAS
CSCD
北大核心
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)
关键词
卡拉库尔羊TNF-α
表达纯化
抗原性分析
Karakul sheep TNF -α
tumor necrosis factor alpha
expression and purification
antigenicity analysis
