分泌型磷脂酶A_2cDNA与载体pRc/CMV定向克隆及表达  

ORIENTATION CLONING OF sPLA-pRc/CMV AND THE EXPRESSION OF sPLA

作　　者：傅桂莲[1] 盖晓东[2] 刘华欣 

机构地区：[1]吉林医学院实验研究中心,132001 [2]吉林医学院病理教研室 [3]吉林空军高等专科学校

出　　处：《中国现代医学杂志》1999年第11期3-4,共2页China Journal of Modern Medicine

摘　　要：为了构建分泌型磷脂酶Ａ２（ｓｅｃｒｅｔａｒｙ ｐｈｏｓｐｈｏｌｉｐａｓｅ Ａ２，ｓＰＬＡ２） ｃＤＮＡ的有效表达系统，该文从无效表达的重组体ｓＰＬＡ２ － ｐＧＥＭ７ 中制备ｓＰＬＡ２ 片段，将其插入中间载体ＢｌｕｅｓｃｒｉｐｔＢＳＳＫＲ的ＥｃｏＲ１ 位点，利用ｓＰＬＡ２－ ＢＳＳＫ重组体转化ＤＨ１ 感受态细胞，筛选ａｎｔｉｓｅｎｓｅｓＰＬＡ２－ ＢＳＳＫ 重组体，应用Ｘｂａｌ、Ｈｉｎｄ３ 双酶消化该重组体并回收带有不同粘性末端的ｓＰＬＡ２ 片段，因Ｘｂａｌ、Ｈｉｎｄ３ 消化载体ｐＲｃ／ＣＭＶ，使之线性化并带有Ｘｂａｌ、Ｈｉｎｄ３ 的粘性末端。ｓＰＬＡ２ 定向插入ｐＲｃ／ＣＭＶ 载体，构建成具有ＣＭＶ 强启动子的表达系统。重组体转染鼠巨噬细胞，扩增培养，从细胞中提取总ＲＮＡ，Ｎｏｒｔｈｅｒｎ 杂交法检测ｓＰＬＡ２ ｍＲＮＡ。结果表明ｐＲｃ／ＣＭＶ 可以用于ｓＰＬＡ２ 表达及各种因素影响表达的研究。To construct an effective expression system for secretory phospholipase A2 (sPLA2) cDNA,we got the sPLA2 cDNA from pGEM7 vector which couldn't express sPLA2 mRNA,and then inserted sPLA2 fragment into the second vector Bluescript BSSK in EcoRl site.We transfered sPLA2-BSSK recombinant into DH1,screened antisense sPLA2-BSSK,digested it by Xbal,HindⅢ and recovered sPLA2 fragment with different cohesive ends.We also cut the vector pRc/CMV with two enzymes to make pRc/CMV be lineared.After cloning sPLA2 into pRc/CMV,the sPLA2-pRc/CMV recombinant was transfected into macrophage of mouse.The cells of mouse were multiplied and RNA was isolated,mRNA of sPLA2 was checked with Northern Blot.The results showed that pRc/CMV was an effective vector for expression study of sPLA2.

关 键 词：磷脂酶A2 定向克隆 NORTHERN杂交 

分 类 号：Q78[生物学—分子生物学] Q556.1