恙虫病东方体Karp株核酸疫苗的构建及其免疫功能初步研究

Construction of Orientia tsutsugamushi Strain Karp DNA Vaccine and Primary Study of Its Immune Function

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 Orientia tsutsugamushi DNA vaccine containing the Sta56 gene and study the immune responses. Methods The target fragment containing the whole ORF of the StaS6 gene was generated by double digesting pMD18/StaS6 plasmid DNA, and sub-cloned into the plasmid pVAX1 to produce the recombinant plasmid pVAXl-StaS6. The recombinant plasmid was then tiansfected into Hela cell. The StaS6 expression was detected by western blot. Transfected L929 cell was infected by Orientia,tsutsugamushi. The numbers of orientia in the cells were counted by Ciemsa stain to indicate the cellular immunity phenomenon induced by recombinant plasmid. Results The recombinant plasmid pVAXl-StaS6 containing the correct StaS6 ORF was expressed in Hela cell. The recombinant Sta56 protein in the culture suspension could be detected by rabbit antiserum to Orientia tsutsugamushi strain Karp. The amount of orientia in L929 cell transfected by pVAX1-Sta56 decreased remarkably compared with the control cell transfected pVAX1 only,showing the induced resistant to the attachment and invasion of Orientia tsutsugamushi by pVAX1-StaS6. Conclusion The DNA vaccine pVAXl-StaS6 expressing Sta56 surface protein antigen was successfully constructed and it could induce cellular immunity in transfected cells.