摘要
目的构建结核分枝杆菌蛋白Ag85A/MPT-64融合基因为基础的DNA疫苗,并利用ELISPOT技术检测其免疫原性。方法利用PCR和基因克隆技术从结核分枝杆菌H37Rv基因组中扩增出Ag85A/MPT-64编码基因,构建真核表达载体,用酶切和双向DNA测序进行鉴定。鉴定正确的阳性克隆重组质粒,肌注免疫小鼠,3周后用ELISPOT法检测抗体滴度。结果 构建到真核载体中的结核分枝杆菌H37Rv株Ag85A/MPT-64融合基因经序列测定证实无突变。ELISPOT法检测几何平均滴度为1∶1 000。结论以Ag85A/MPT-64融合基因为基础的DNA疫苗的成功构建和表达以及ELISPOT技术的应用,为进一步研究其在结核病与肿瘤防治方面的作用奠定了基础。
Objective To construct DNA vaccine based on Ag85A/MPT-64 gene of Mycobacterium tuberculosis and analyze the immunogenicity by ELISPOT assay. Methods The gene encoding Ag85A and MPT-64 protein was amplified by PCR from the genome of Mycobacterium tuberculosis H37Rv strain,and cloned into eukaryotic expressing vector and were confirmed by restriction endonuclease digestion and bidirectional DNA sequencing. Then the recombinant plasmid of the correct clone was injected to mice. 3 weeks later titers of serum antibody against Ag85A/MPT-64 were detected by ELISPOT assay. Results The nucleotide sequencing of the recombinant gene encoding Ag85A/MPT-64 of Mycobacterium tuberculosis H37Rv strain has no mutation and the gene was correctly inserted into the vector as confirmed by double restriction endonuclease digestion. Mean geometric antibody titer of Ag85A/MPT-64 was 1 ： 1 000 by ELISPOT assay. Conclusion DNA vaccine constructed and expressed successfully based on the gene encoding Ag85A/ MPT-64 fusion protein of Mycobacterium tuberculosis and the application of the ELISPOT assay is a foundation for further research into the prevention and treatment of tuberculosis and carcinoma.
出处
《中国实验诊断学》
2012年第1期20-23,共4页
Chinese Journal of Laboratory Diagnosis
基金
吉林省自然科学基金项目(编号:201115079200505115)
