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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 ,TIG_310的表达量逐渐恢复正常
OBJECTIVE: To study the prokaryotic expression, cellular and subcellular localization of Ty2 1a immunostaining gene (TIG_310) and its variation in Ty2 1a immunization. Methods: The possible coding region of TIG_310 was amplified by PCR, cloned into pQE30 vector and induced in M15 strain. The in situ hybridization was used to locate the TIG_310 gene in intestinal mucosa. Subcloned into the green fluorescent protein expression vector pEGFP_N1, transfected into L cells by electroporation to determine its subcellular localization; RT_PCR study of its changes in Ty2 1a immunization. RESULTS AND CONCLUSION: The coding region of TIG_310 was expressed in prokaryotic expression system. The result of in situ hybridization showed that the gene was expressed in epithelial cells of intestinal mucosa. The results of fusion with green fluorescent protein showed that the protein localized in the whole in the cell. The gene was overexpressed in the gastrointestinal mucosa of normal BALB / c mice, and the expression of TIG_310 was significantly increased after the first intragastric administration of Ty2 1a. With the increase in the number of immunization, the expression of TIG_310 gradually returned to normal