旋毛虫脱氧核糖核酸酶Ⅱ蛋白的制备及其定位研究

Preparation and Localization of Deoxyribonuclease Ⅱ Protein from Trichinella spiralis

为获得旋毛虫(T1)脱氧核糖核酸酶Ⅱ(DNase Ⅱ)蛋白,观察其在肌幼虫虫体中的分布,以旋毛虫(T1)肌幼虫cDNA为模板,利用RT-PCR技术扩增旋毛虫脱氧核糖核酸酶Ⅱ(TsDNase Ⅱ)蛋白的编码基因tsdnase Ⅱ,将其克隆到原核表达载体pCold-TF中,转化至大肠杆菌BL21(DE3)感受态细胞中,以IPTG诱导表达,表达产物进行SDS-PAGE电泳分析;利用酶联免疫吸附试验(ELISA)及Western blot分析重组Ts DNaseⅡ(rTsDNase Ⅱ)蛋白的反应原性;将rTsDNase Ⅱ蛋白免疫6-8周BALB/c小鼠,制备其多克隆抗体;制作旋毛虫肌幼虫冰冻切片,利用免疫荧光试验观察TsDNase Ⅱ蛋白在虫体中的分布。结果,成功地扩增出了长度为942 bp的旋毛虫tsdnase Ⅱ基因片段,构建了其原核表达质粒pCold-TF-tsdnase Ⅱ;转化了重组表达质粒的大肠杆菌BL21(DE3)成功可溶表达了rTsDNase Ⅱ,大小为86 kDa;ELISA与Western blot分析结果显示,rTsDNase Ⅱ蛋白能与感染旋毛虫的小鼠血清发生特异性反应;冰冻切片免疫荧光分析显示,TsDNase Ⅱ分布于旋毛虫肌幼虫整个虫体。研究结果为进一步分析TsDNase Ⅱ的特性与功能、利用该蛋白研发旋毛虫病新型防控策略打下了基础。

In order to obtain the deoxyribonuclease Ⅱ(DNase Ⅱ)protein of Trichinellaspiralis(T1)and observe its distribution in T.spiralis muscle larvae,tsdnaseIIgene was amplified by RT-PCR using T.spiralis muscle larva cDNA as template and cloned into prokaryotic expression vector pCold-TF.The recombinant plasmid was then transformed intoEscherichiacoliBL21(DE3)competent cells and induced by IPTG.The expressed product was analyzed by SDS-PAGE electrophoresis.The reactogenicity of recombinant TsDNase Ⅱ(rTsDNaseⅡ)protein was analyzed by ELISA and Western blot.BALB/c mice aged 6-8 weeks were immunized with rTsDNase Ⅱ to prepare the polyclonal antibodies.The frozen sections of T.spiralis muscle larvae were analyzed by immunofluorescence assay to observe the subcellular distribution of TsDNase II.The results showed that the tsdnaseIIgene fragment was successfully amplified and its length was 942 bp.The prokaryotic expression plasmid pCold-TF-tsdnase Ⅱ was successfully constructed and theE.coliBL21(DE3)transforming with the recombinant expression plasmid successfully expressed the rTsDNase Ⅱ,which was about 86 kDa.ELISA and Western blot analysis showed that the rTsDNase Ⅱ protein could specifically react with the sera of mice infected with T.spiralis.The frozen sections ofT.spiralismuscle larvae were analyzed by immunofluorescence and the results showed that TsDNase Ⅱ was distributed on the throughout body of T.spiralismuscle larvae.These results laid a foundation for further analysis of the characteristics and functions of TsDNase Ⅱ,as well as the development of new prevention and control strategies for trichinellosis.