摘要
目的克隆、表达与纯化结核分枝杆菌CFP32蛋白,并对其进行细胞免疫学特性评价。方法 PCR扩增cfp32基因片段,与pET-30a(+)构建重组表达质粒,在大肠杆菌中诱导表达,对融合蛋白进行纯化,ELISPOT试验测定其细胞免疫应答,通过牛结核外周血IFN-γ释放试验,评价其作为刺激原的能力。结果构建了正确基因序列的重组质粒,并在BL21(DE3)中高效可溶性表达,融合蛋白大小为32ku,纯度达91.8%。ELISPOT显示CFP32蛋白刺激机体分泌IFN-γ和IL-4的细胞数相当,呈现Th1/Th2的平衡。在牛结核外周血IFN-γ释放试验中,其阳性符合率和阴性符合率分别为40%和73.3%,与牛型PPD刺激结果的相关系数为0.684。结论成功构建CFP32蛋白重组表达菌,该蛋白引起的免疫应答趋向于Th1/Th2的平衡,并且有作为刺激原用于牛结核病检测的潜力。
The CFP32 protein of Mycobacterium tuberculosis was expressed in E. coli BI.2 （DE3） and its cellular immu noproperties was analysed. The gene cfp32 was amplified by polymerase chain reaction （PCR） and was inserted to vector pET- 30a（＋） to construct the recombinant plasmid after sequence analysis. Then expression of CFP32 with His tag in E. coli BL21 （DE3） was detected through SDS-PAGE and Western blot assay, The relative molecular mass of this protein was about 32 ku. IFN-γ specific secreting cell spots as well as IL-4 were detected by ELISPOT assay. The positive and negative coincidence rate of CFP32 in the BOVIGAM^TM Mycobacteriurn boris gamma interferon test kit for cattle were 40% and 73.3%, respectively. The correlation between bovine PPD and the CFP32 protein was 0. 684. CFP32 protein was expressed successfully and could in duee Th1/Th2 eytokine balance in mice. These results show a potential use of CFP32 protein for bovine tuberculosis diagnosis.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2014年第6期578-582,共5页
Chinese Journal of Zoonoses
基金
国家"973"项目(No.2012CB518805)
国家公益性行业科研专项(No.200903027)
江苏省农业三新工程(No.SXGC[2012]402)
江苏高校优势学科建设工程资助项目联合资助~~
