人碱性成纤维细胞生长因子cDNA的克隆及在毕赤酵母中的表达

目的：克隆和表达人碱性成纤维细胞生长因子(hbFGF)，以便进一步研究其功能。方法：从人神经胶质瘤细胞中提取总RNA，采用RT-PCR方法，扩增出hbFGF cDNA片段，与pMD18-T载体连接后作DNA序列分析，并用亚克隆的方法构建pPICZα-hbfFGF，将测序正确的重组质粒用SacI线性化后电转化至毕赤酵母X-33，经PCR法、SDS-PAGE和Western blotting分析表达产物,从而筛选出能够稳定、高效分泌表达hbFGF的酵母工程菌。结果：经测序证实获得的hbFGF序列与GenBank（登录号NM002006）报道的完全一致，甲醇诱导表达后培养液上清的SDS-PAGE凝胶电泳显示在相对分子质量约18 000有一蛋白条带，Western blotting结果显示表达产物与抗人碱性成纤维细胞生长因子抗体产生特异性条带，证明rhbFGF蛋白的表达。结论：成功克隆了hbFGF 基因，并在毕赤酵母体系中进行了表达。

Cloning of hbFGF cDNA and expression of recombinant hbFGF in Pichia pastoris

Objective To clone and express human basic fibroblast growth factor(hbFGF) and investigate its function.Methods The total RNA was isolated from human glioma cells and hbFGF was cloned by RT-PCR.After the PCR product was ligated with pMD18-T and sequenced,the pPICZα-hbFGF recombinant plasmid was constructed by subclone and transformed into Pichia pastoris X-33 via electroporation which was linearized with SacI.The fusion protein was verified and the transformant Pichia pastoris with high effective expression of hbFGF was screened by using PCR,SDS-PAGE and Western blotting.Results The obtained hbFGF gene was consistent with the hbFGF reported in GenBank(Accession no.NM002006).The hbFGF was expressed and secreted in X-33.SDS-PAGE and Western blotting analysis showed that the expressed protein in the culture supernatant induced by methanol was approximately 18 000 and could react specifically with anti-hbFGF antibody.Conclusion hbFGF gene is cloned successfully and expressed in Pichia pastoris.