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目的构建恙虫病东方体Karp株Sta56抗原候选核酸疫苗并初步探讨其可能诱导的免疫功能。方法从连有恙虫病东方体Karp株编码56000u表膜蛋白基因开放读码框全长的T载体质粒pMD18/Sta56中双酶切出目的基因,定向亚克隆构建核酸疫苗质粒载体pVAX1-Sta56,转染Hela细胞,WesternBlot分析Sta56蛋白的表达及其免疫原性,转染L929细胞,接种恙虫病东方体,Giemsa染色细胞内恙虫病东方体数目比较,初步探讨pVAX1-Sta56可能诱导的细胞免疫现象。结果pVAX1-Sta56连有正确读码框架的Sta56全长基因。转染有pVAX1-Sta56的Hela细胞培养上清可检测到被兔抗恙虫病东方体Karp株抗血清识别的特异条带。转染有pVAX1-Sta56的L929细胞内恙虫病东方体数目显著少于转染pVAX1的L929细胞(P<0.01)。结论成功构建能够表达Sta56表膜蛋白抗原的核酸疫苗载体pVAX1-Sta56,并能诱导产生细胞免疫功能。
Objective To construct Sta56 antigen candidate nucleic acid vaccine of Orientia tsutsugamushi Karp Strain and to explore its possible immune function. Methods The target gene was double-digested by T vector plasmid pMD18 / Sta56 with the open reading frame of 56000u, which was encoded by Orien tsugustae tsutsugamushi Karp strain. The target gene was subcloned by directional subcloning to construct the DNA vaccine plasmid vector pVAX1-Sta56 The expression of Sta56 protein and its immunogenicity were analyzed by Western Blot, and the number of Oriental body of scrub typhus was detected in L929 cells transfected with L929 cells. Results The full-length Sta56 gene of pVAX1-Sta56 with correct reading frame was obtained. The Hela cell culture supernatant transfected with pVAX1-Sta56 could detect the specific band recognized by the antiserum against the Karp strain of T. tsutsugamushi. The number of oriental tsutsugamushi in L929 cells transfected with pVAX1-Sta56 was significantly less than that in L929 cells transfected with pVAX1 (P <0.01). Conclusion The nucleic acid vaccine vector pVAX1-Sta56, which can express Sta56 surface antigen, was successfully constructed and could induce cellular immune function.