查菲埃立克体120 000表面抗原蛋白基因的克隆与表达

目的 构建查菲埃立克特异性表面抗原P120基因的原核表达质粒，并使该基因在E.coli中高效表达。方法 依据查菲埃立克体P120基因序列设计一对特异性引物，用PCR从查菲埃立体克体的基因中扩增含有编码P120原决定簇的基因片段，而后将PCR产物用T载体克隆。用酶将PCR产物从T载体切下，将它定向插入原核表达质粒PET11C构建PET11C／p120基因。PET12C／p120重组质粒转化的E．coli在TPTG的诱导下产生一分子量为41000的天然蛋白，它的产量占细菌蛋白总量的28％，用免疫印迹分析证明它能特异地与查菲埃立体克感染病人血清反应。结论 查菲埃立克体特异性的P120抗原在原核表达系统被高效表达，该抗原的研制成功为制备人单核细胞埃立克体病诊断抗原和疫苗奠定了基础。

Cloning and expression of 120 000 surface antigen of Ehrlichia chaffeensis

Objective To construct a prokaryotic expression plasmid with a gene encoding a 120 000 surface antigen (P120) of Ehrlichia chaffeensis for its highly express in the E. coli cells. Methods The DNA fragment containing gene encoding the immunodominant of P120 was am- plifyed with a pair of primers designed based on the reported sequence of the p120 gene by PCR. The PCR product was cloned with T-vector and the cloned DNA fragment was cut off from the T-vector with NdeI and BamHI. The DNA fragment was ligated into an expression plasmid (PET11C) and transformed the E. coli BL21(DE3) with the recombinant plasmid (PET11C/p120). The expression of p120 gene in the E. coli was induced by IPTG. Results The DNA fragment amplified by PCR was 1 119 bp and was identified to be the p120 gene of E. chaffeensis by analyzing its se- quence. A 41 000 natural protein that was 28% of the total protein of the E. coli was identified in this transformed E. coli by SDS-PAGE and it reacted specifically with the sera of patients infected by E. chaffeensis in Western blotting analysis. Conclusion The E. coli transformed by the recombinant plasmid (PET11C/p120) yield a high level of P120 antigen of Ehrlichia Chaffeensis, which lays a foundation of preparing a large amount of P120 antigen for serological diagnosis and vaccination of human monocytic ehlichiosis.