pCDR1 Th表位和CTLA4Ig双基因共表达真核表达载体的构建

Construction of eukaryotic expression vectors stably expressing pCDR1 Th epitope and CTLA4Ig fusion gene

目的构建能稳定表达pCDR1 Th表位和CTLA4Ig融合基因的真核表达载体,探讨其在小鼠体内的表达并对表达产物进行鉴定。方法用Touchdown PCR法扩增CTLA4Ig基因,同时引入pCDR1Th表位。将PCR产物连接真核表达载体pcDNA3.1(+),构建pcDNA3.1(+)-CTLA4Ig-pCDR1。将构建表达CTLA4Ig-pCDR1减毒鼠伤寒沙门菌SL7207喂饲BALB/c小鼠,取脾脏进行免疫组化鉴定重组蛋白在动物体内的表达。结果酶切鉴定和基因序列测定显示重组质粒构建成功。重组蛋白在BALB/c小鼠脾脏免疫细胞胞浆中有阳性表达。结论成功构建了能稳定表达pCDR1 Th表位和CTLA4Ig融合基因的真核表达载体。

Objective To construct an eukaryotic expression vector stably expressing pCDR1 Th epitope and CTLA4Ig fusion gene,and to identify its expression product. Methods Touchdown PCR was used to amplify CTLA4Ig cDNA and introduce pCDR1 Th epitope. The PCR product was ligated into the multiple clone site of eukaryotic expression vector pcDNA3. 1 （ ＋ ） by gene recombination technique. After the attenuated Salmonella Typhimurium SL7207 containing the recombinant expression plasmids pcDNA3.1 （＋） CTLA4Ig pCDR1 immunized BALB/c mice orally,spleens were removed to identify the recombinant proteins expres sion by immunohistochemistry assay. Results Restriction analysis and DNA sequence analysis showed that the pCDR1 Th epitope and CTLA4Ig cDNA had been successfully inserted into pcDNA3.1（＋） eukaryotic expression vector. The fusion protein could be detected in the cytoplasm of spleen cells. Conclusion The eukaryotic expression vector stably expressing pCDR1 Th epitope and CTLA4Ig fusion gene is successfully constructed.