凝血因子Ⅷ C2结构域表达载体的构建及其在真核细胞NIH3T3中的表达  

Construction of recombinant vector containing FⅧ-C2 and its expression in NIH3T3 cells

作　　者：刘晓冬[1] 马淑梅[1] LANG Glen SEHON Alec 

机构地区：[1]吉林大学公共卫生学院卫生部放射生物学重点实验室,吉林长春130021 [2]加拿大曼尼拖巴大学医学院免疫系

出　　处：《吉林大学学报（医学版）》2005年第1期35-38,63,共5页Journal of Jilin University：Medicine Edition

基　　金：中国博士后科学基金资助课题 (2 0 0 3)

摘　　要：目的 :构建人凝血因子 F 中具有免疫活性的 C2结构域表达载体并通过真核宿主细胞表达出多肽。方法 :从 HEK2 93细胞提取总 RNA,经 RT- PCR扩增获得目的片段。将该片段与信号肽共同构建在同一表达载体 ,并在 NIH 3T3细胞中表达 ,Western blotting检测。结果 :经限制性酶切鉴定和测序分析证实 ,采用 RT- PCR成功地完成 F - C2 c DNA的扩增和表达载体的构建 ,重组载体转染进入 NIH 3T3细胞后 ,经检测培养上清中的蛋白成分证实 ,成功地表达出 F - C2结构域多肽。结论 :重组表达载体的构建是表达出分泌型 F C2多肽的一种可行的方法。Objective To construct the complementary DNA (cDNA) of FⅧ-C2 by gene engineering and express the biologically active domain in NIH 3T3. Methods The total RNA extracted from 293 cells and FⅧ-C2 was amplified by RT-PCR. The product was then inserted into pTARGET vector together with signal peptide sequence. The recombinant vector was transfected into NIH3T3 cells followed by the detection of the expression of FⅧ-C2 by Western blotting. Results The cDNA of FⅧ-C2 was identified correctly by endonucleases cleavage and sequencing. The recombinant FⅧ-C2 peptide was obtained by detection of FⅧ-C2 from the supernatant of NIH3T3. Conclusion The construction of recombinant vector is a feasible way to express secretive FⅧ-C2.

关 键 词：因子Ⅷ C2结构域 逆转录聚合酶链反应 基因重组 

分 类 号：Q78[生物学—分子生物学]