摘要
目的将核小体Th表位与CTLA4Ig融合基因融合,研究CTLA4Ig作为真核表达载体的可行性。方法用touchdownPCR法扩增CTLA4Ig基因,同时引入核小体Th表位(H2B14-28)。将PCR产物连接真核表达载体pcDNA3.1穴+雪,构建pcDNA3.1穴+雪-CTLA4Ig-H2B。将表达质粒转染COS-7细胞,Westernblot检测转染细胞裂解上清中融合蛋白的表达。将构建表达CTLA4Ig-H2B的减毒鼠伤寒沙门氏菌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。在pcDNA3.1穴+雪-CTLA4Ig-H2B质粒转染后48h细胞裂解上清中,检测到CTLA4Ig融合蛋白的表达,该蛋白能与抗人CTLA-4单抗特异结合。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核小体Th表位和CTLA4Ig融合基因的真核表达载体。
Objective To study the feasibility of CTLA4Ig as eukaryotic expression vector by fusing nucleosome Th epitope in CTLA4Ig eDNA. Methods Touchdown PCR was used to amplify CTLA4Ig eDNA and introduce nucleosome Th epitope. The PCR product was ligated into the multiple clone site of eukaryotic expression vector peDNA3.1 （＋） by gene recombination technique. Then the recombinated plasmid pcDNA3.1 （＋）-CTLA4Ig-H2B was transfeeted into COS-7 cells using DOTAP. The expression of fusion protein in the supernatant of the cell disruption was detected with Western blotting. After the attenuated Salmonella Typhimurium SL7207 containing the recombinant expression plasmids peDNA3.1 （＋）-CTLA4Ig-H2B immunized BALB/c mice orally,spleens were removed to identify the recombinant proteins expression by immunohistoehemistry. Results Restriction analysis and DNA sequence analysis showed that the nueleosome Th epitope and CTLA4Ig eDNA had been successfully inserted into pcDNA3.1 （＋） eukaryotic expression vector. The fusion protein could be detected in the superuatant of cell disruption 48 h after the transfection of peDNA3.1（＋）-CTLA4Ig-H2B. This protein specifically bound with human CTLA--4 monoelonal antibody. The fusion protein could be detected in the the cytoplasm of spleen cells. Conclusion The eukaryotic expression vector containing nucleosome Th epitope and CTLA4Ig gene is successfully constructed.
出处
《生物医学工程与临床》
CAS
2006年第6期387-390,F0003,共5页
Biomedical Engineering and Clinical Medicine
基金
国家自然科学基金(30200258)
