恙虫病东方体合肥分离株截短56kDa蛋白抗原表达及活性分析

ANTIGEN EXPRESSION AND IMMUNE ACTIVITY ANALYSIS OF TRUNCATED 56 kDa PROTEIN DERIVED FROM THE ISOLATED HEFEI STRAIN OF ORIENTIA TSUTSUGAMUSHI

本文获得恙虫病东方体合肥分离株截短56kDa外膜蛋白基因工程纯化抗原，并鉴定其免疫学活性，为进一步研制广谱诊断试剂、疫苗以及分析其在东方体致病的分子机制奠定基础。我们通过DNA Star和Mega等软件对含有440个氨基酸的合肥株56kDa蛋白序列进行抗原性分析；将合肥株56kDa截短蛋白基因克隆至原核表达载体pET28a获重组质粒，转入大肠杆菌E．coli BL21感受态中，经IPTG诱导，SDS—PAGE电泳分析检测蛋白表达；采用亲和层析法纯化目的蛋白，Western-blot及ELISA法鉴定其免疫学活性。序列分析发现合肥分离株56kDa蛋白可分为4个保守区和3个可变区，抗原性分析结果显示，其保守区的亲水性和抗原性均有很高峰值。SDS—PAGE电泳显示约56kDa的目的重组蛋白表达条带，Western—blot分析显示该重组蛋白可与恙虫病病人血清反应，阴性血清则未出现相应印迹，ELISA分析结果显示该蛋白最低浓度为80ng／μL时仍可检出阳性血清；5μg／mL蛋白可检测阳性血清滴度高达1：12800，证实其具有较强反应原性。本实验获得恙虫病东方体合肥株56kDa截短外膜蛋白基因工程抗原具有良好的抗原活性，有望进一步应用于恙虫病的诊断、疫苗研制及致病机制分析。

This study is an attempt to obtain purified antigen of truncated 56 kDa outer membrane protein derived from the isolated Hefei strain of Orientia tsutsugamushi, and identify its immunological activity, which may benefit the further development of diagnosis reagent with broad spectrum, vaccine development and the illustration of related molecular pathogenesis of O. tsutsugamushi. Two softwares, DNAStar and Mega, were used to analyze the antigenicity of truncated Hefei 56 kDa protein sequence with 440 amino acids. The truncated Hefei 56 kDa protein gene was cloned into prokaryotic expression plasmid pET28a, and then expressed in E. coli BL21 induced by IPTG. The expressed protein was separated and analyzed by SDS-PAGE. The protein was purified by affinity chromatography and its immunological activity was identified by Western blotting and ELISA. The results show that the truncated 56 kDa protein includes four conserved regions and three variable regions, which shows high hydrophilicity and antigenicity. SDS-PAGE showed the expressed fusional protein on the gel at about 55kDa of molecular mass. The recombinant protein can be recognized by patient＇s positive serum by western blot while the negative serum cannot result in the same blot band. ELISA results indicated that the recombinant proteins showed good antigenicity that can distinguish the positive and negative serum successfully. The results suggest that the purified recombinant protein of Hefei 56 kDa with immuno-reactivity may be a promising diagnostic antigen for the development of the diagnosis kit for tsutsugamushi disease, the vaccine development and the illustration of pathogenic mechanism of O. tsutsugamushi.