重叠区扩增基因拼接法在构建膜锚定共刺激分子融合基因上的应用
易平勇,余海,马文学,孙文军,姜槐
摘要(Abstract)：
目的 将小鼠共刺激分子 B7- 1(m B7- 1)基因的胞外区与人胎盘碱性磷酸酶 (h PL AP- 1) C末端信号肽序列进行拼接 ,探讨其形成融合基因的作用。方法 利用重叠区扩增基因拼接法 ,分别扩增 h PL AP- 1C末端信号肽序列和 m B7- 1基因的胞外区序列 ,然后将二段基因拼接起来 ,拼接后与 p GEM- T克隆载体连接 ,酶切和测序鉴定。结果 分别成功扩增出 h PL AP- 1C末端信号肽序列 15 5 bp和 m B7- 1基因的胞外区序列 76 4 bp,并将二段基因序列成功拼接 (891bp) ,经测序是正确的。结论 重叠区扩增基因拼接法能拼接 m B7- 1基因的胞外区与 h PL AP-1C末端信号肽序列 ,能形成融合基因
关键词(KeyWords)： 融合蛋白质类;刺激抗原;碱性磷酸酶;胎盘;糖基磷脂酰肌醇类;重叠区扩增基因拼接法
基金项目(Foundation):
作者(Author): 易平勇,余海,马文学,孙文军,姜槐
参考文献(References)：
- [1] 张学敏,王宜强.靶向新基因的分子克隆策略-理论与方法[M].北京:军事医学科学出版社,1999.69-74.
- [2] HortonRM,HuntHD,HoSN,et al.Engineering hy-brid gene without the use of restriction enzymes:genesplicing by overlap extension[J].Gene,1989,77(1):61-68.
- [3] ChenL,McGowanP,AsheS,et al.Tumor im muno-genicity determines the effect ofB7costim ulation onT cell- m ediated tum or imm unity[J].J ExpMed,1994,179(2):523-532.
- [4] 胡锦跃,王 飒,朱建高,等.B7基因修饰的肿瘤细胞诱导抗大肠癌主动免疫[J].中国肿瘤临床,2000,27(2):137-141.
- [5] 颜子颖,王海林.精编分子生物学实验指南[M].第一版,北京:科学出版社,1999.35-36.
- [6] MillanJL.MolecularCloning andSequenceAnalysisofHum anPlacentalAlkalinePhosphatase[J].J BiolChem,1986,261(7):3112-3115.
- [7] FreemanGJ,GrayGS,Gim miCD,et al.Structure,Expression,andT CellCostimulatoryActivity of theMurineHomologue of theHumanB L ymphocyteActi-vationAntigenB7[J].JExpMed,1991,174(3):625-631.
- [8] 胡福泉,扬 恬.现代基因操作技术[M].北京:人民军医出版社,2000.157-165.
- [9] 金冬雁,黎孟枫.分子克隆实验指南[M].第二版,北京:科学出版社,1992.1-69.
- [10] TownsendSE,SuFW,AthertonJM,et al.Speci-ficity andL ongevity ofAntitum orImm uneRespons-esInduced byB7- transfectedTum ors[J].CancerRes,1994,54(24):6477-6483.
- [11] TykocinskiML,KaplanDR,MedofME.Antigen-PresentingCellEngineering[J].AJP January,1996,148(1):1-16.
- [12] BergerJ,HowardAD,BrinkL,et al.COOH- termi-nalRequirements for theCorrectProcessing of aPhosphatidylinositol-GlycanAnchoredMem braneProtein[J].J BiolChem,1988,263(20):10016-10021.
- [13] OdaK,ChengJ,SakuT,et al.Conversion of secreto-ry proteins into m em brane proteins by fusing with aglycosylphosphatidylinositol anchor signal of alkalinephosphatase[J].BiochemJ,1994,301(2):577-583.
- [14] MedofME,NagarajanS,TykocinskiML.Cell- sur-face engineering withGPI- anchored proteins[J].FASEB J,1996,10(5):574-586.