摘要
目的 :研究Ty2 1a免疫相关新基因 (Ty2 1aimmunization_associatedgene ,TIG_310 )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TIG_310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TIG_310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 :TIG_310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 。
Objective:To investigate the expression, cellular and subcellular localization of the novel mouse Ty21a immunization_associated gene (TIG_310) and to find out its variation during the Ty21a immunization. Methods:The coding region of TIG_310 was gained by PCR and was then inserted into pQE30 vector. The expression of TIG_310 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 TIG_310, 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 TIG_310 expression during the Ty21a immunization was obtained by RT_PCR. Results and Conclusions:The TIG_310 gene was successfully expressed in E.coli M15. The expressed protein existed in the inclusion body. In situ analysis showed that the TIG_310 gene was mainly in the epithelial cells of the gastrointestinal tract. The protein coded by TIG_310 gene distributed through out the cells. The expression of TIG_310 was elevated after Ty21a immunization.
出处
《军事医学科学院院刊》
CSCD
北大核心
2003年第2期99-102,共4页
Bulletin of the Academy of Military Medical Sciences
基金
国家"973"计划资助项目 (G19990 5 41_0 3 )
关键词
黏膜免疫系统
Ty21α疫苗
TIG-310基因
基因表达
细胞定位
亚细胞定位
Ty21a immunization_associated gene
cellular localization
subcellular localization
prokaryotic expression
gene expression
polymerase chain reaction
