【摘要】 目的 :研究Ty2 1a免疫相关新基因 （Ty2 1aimmunization_associatedgene ,TIG₃10 ）的原核表达、细胞和亚细胞定位及其在Ty2 1a免疫中的变化规律。方法 :采用PCR技术 ,扩增TIG₃10可能的编码区 ,将其克隆至pQE30载体 ,在M15菌株中进行诱导表达 ;采用原位杂交技术对TIG₃10基因的表达产物在肠黏膜组织进行细胞定位 ;将其亚克隆至绿色荧光蛋白表达载体pEGFP_N1,通过电击转染导入L细胞 ,确定其亚细胞定位 ;通过RT_PCR研究其在Ty2 1a免疫中的变化规律。结果与结论 :TIG₃10的编码区在原核表达系统中获得包涵体表达 ;原位杂交结果显示该基因表达在肠黏膜的上皮细胞中 ;与绿色荧光蛋白融合表达的实验结果表明 ,该蛋白定位于整个细胞内。该基因在正常的BALB/c小鼠的胃肠黏膜高表达 ,在Ty2 1a第一次灌胃免疫后表达量显著增高 ,随着免疫次数的增加 ,TIG₃10的表达量逐渐恢复正常更多还原

【Abstract】 Objective:To investigate the expression, cellular and subcellular localization of the novel mouse Ty21a immunization_associated gene （TIG₃10） and to find out its variation during the Ty21a immunization. Methods:The coding region of TIG₃10 was gained by PCR and was then inserted into pQE30 vector. The expression of TIG₃10 in E.coli M15 was induced by IPTG at 37℃and was then analyzed by SDS_PAGE. The cellular localization information was obtained by in situ hybridization. To obtain the subcellular localization of TIG₃10, the gene was subcloned into the pEGFP_N1 vector and then transfected into L cells. The expression of fusion protein was analyzed by confocal microscopy. The information about TIG₃10 expression during the Ty21a immunization was obtained by RT_PCR. Results and Conclusions:The TIG₃10 gene was successfully expressed in E.coli M15. The expressed protein existed in the inclusion body. In situ analysis showed that the TIG₃10 gene was mainly in the epithelial cells of the gastrointestinal tract. The protein coded by TIG₃10 gene distributed through out the cells. The expression of TIG₃10 was elevated after Ty21a immunization.更多还原