鼠伤寒沙门菌SseK1蛋白的表达及其免疫生物学特性研究

Expression purification and immunological characteristics of Salmonella typhimurium antigen SseK1

目的:研究鼠伤寒沙门菌SseK1蛋白免疫生物学特性及其作为潜在免疫抗原蛋白的可能性。方法:利用PCR方法从鼠伤寒沙门菌SL1344株克隆获得sseK1基因片段,测序分析正确后连接至原核表达载体pET-32a(+),并在大肠杆菌BL21(DE3)感受态中诱导表达。Western blot进一步检测镍离子亲和层析法纯化的SseK1蛋白并进行免疫生物学特性分析。结果:PCR及测序结果表明BL21(pET-32a-SseK1)构建成功。经SDS-PAGE分析显示,表达的SseK1蛋白大小约为43 kD,在原核表达反应器中主要以可溶性形式存在于菌体裂解上清中;经Western blot分析显示,SseK1蛋白可被鼠伤寒沙门菌阳性多抗血清识别,具有良好的反应原性;用SseK1蛋白免疫6周龄BALB/c小鼠,二免后21 d间接ELISA检测的抗体效价高达1∶124000,具有良好的免疫原性。淋巴细胞增殖试验结果表明SseK1能够显著刺激小鼠T淋巴细胞增殖,激发机体细胞免疫应答,差异具有统计学意义(P<0.05)。小鼠攻毒及免疫保护试验表明,鼠伤寒沙门菌强毒株对二免14 d后的小鼠攻毒显示60%以上的免疫保护力。结论:本研究表达的SseK1蛋白具有良好的抗原性,为深入研究鼠伤寒沙门菌与机体在感染和免疫方面的相互作用奠定了一定的理论基础。

Objective:To study the immunological characteristics of Salmonella typhimurium SseK1 protein and its potential as immunizing antigen.Methods:The sseK1 gene was amplified from the Salmonella typhimurium SL1344 strain by PCR.The sequence was confirmed by sequencing and cloned into pET-32 a(+)vector for expression in BL21(DE3)cells.The SseK1 protein was purified by Ni-NTA affinity chromatography and further examined by Western blot and analyzed for immunobiology.Results:Protein SseK1 prokaryotically expressed in this study was mainly present in soluble form in the lysate supernatant of the prokaryotic expression reactor,and the expressed fusion protein size was about 43 kD by SDS-PAGE.The antigenicity of protein SseK1 was demonstrated by specially recognition to Salmonella typhimurium positive polyclonal antibody in Western blot.After SseK1 vaccine was prepared and injected into 6-week BALB/c mice,the antibody titers up to 1∶124000 which was detected with indirect ELISA.The results also showed that significant T lymphopoiesis was detected in the immunized mice,which indicated that SseK1 was able to efficiently induce cellular immunity in the mice(P<0.05).The immunoprotective effect assay showed that the protection rate infected with SL1344 strain of Salmonella typhimurium was more than 60%on the 14 th day post-second immunization.Conclusion:The results showed that the prokaryotically expressed SseK1 in present study shows satisfied antigenicity,and can be used as a antigen protein candidate for recombinant subunit vaccine,which also laid the foundation for further study on the interaction between Salmonella typhimurium and the organism in infection and immunization.