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

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.