【摘要】 目的对田鼠巴贝西虫(Babesia microti)的诊断抗原分泌抗原1(Babesia microtisecreted antigen 1,BmSA1)的截短部分(tBmSA1)进行克隆构建、原核表达与纯化,并对重组tBmSA1进行质谱鉴定以及抗原性鉴定。方法通过PCR扩增截短型tBmSA1基因,将其插入原核表达载体pET-30a(+),转化大肠杆菌BL21(DE3),经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达重组tBmSA1,使用AKTA进行镍亲和层析纯化,并对纯化的重组tBmSA1进行质谱分析,通过Western Blot和ELISA鉴定重组tBmSA1的抗原性。结果成功构建了重组质粒pET-30a(+)-tBmSA1,重组tBmSA1得到高效表达和纯化,质谱分析验证了其正确性,Western Blot和ELISA结果显示其具有较好的抗原性。结论实现了田鼠巴贝西虫tBmSA1的原核高效表达,并验证了其诊断价值,为田鼠巴贝西虫感染诊断奠定了基础。更多还原

【Abstract】 Objective To clone,express and purify Babesia microti truncated secreted antigen 1(tBmSA1)in prokaryotic system;to validate recombinant protein by mass spectrometry(MS)and characterize its antigenicity.Methods tBmSA1 encoded gene was amplified by PCR and cloned into pET-30a(+)vector.The pET-30a(+)-tBmSA1 recombinant plasmid was transformed into E.coli BL21(DE3)for expression by IPTG induction.The recombinant protein was purified by AKTA Nickel-affinity chromatography and confirmed by MS.Its antigenicity was tested by ELISA and Western Blot analysis.ResultsThe recombinant plasmid pET-30a(+)-tBmSA1 was constructed successfully and was effectively expressed.MS analysis showed that the recombinant protein was tBmSA1.The ELISA and Western Blot analysis demonstrated that the recombinant tBmSA1 showed good antigenicity.Conclusion The tBmSA1 gene was cloned and expressed with high efficiency in prokaryotic system successfully,its diagnostic significance was validated,which laid a solid foundation to develop diagnostic assay for Babesia microti.更多还原