人碱性成纤维细胞生长因子真核表达载体的构建及表达

Construction and expression of human bFGF eukaryotic expression vector

目的: 构建研究人碱性成纤维细胞生长因子(bF -GF)真核表达载体,并在体外进行表达和检测.方法: 从骨髓基质干细胞中分离总RNA,进行RT- PCR获得人bFGFcDNA.测序证实其结果正确后,构建真核表达载体.并经BamHⅠ/EcoRⅠ双酶切、PCR及测序鉴定.利用Blast程序,搜索NCBIGenBank中与重组质粒(pVAX1 bFGF)中编码bFGF基因同源的序列.通过脂质体将重组质粒转染入骨髓基质干细胞,使用间接免疫荧光法进行表达产物检测.结果: 经EcoRⅠ/PstBamHⅠ/EcoRⅠ双酶切、PCR及测序鉴定证实,人bFGF基因成功地克隆到真核表达载体pVAX1中;pVAX1- bFGF中编码bFGF的序列与GenBank中所报道的bFGF编码序列完全一致;间接免疫荧光结果显示转染重组质粒的细胞表面有绿色荧光.结论: 成功地构建了人bFGF真核表达载体,其可以在体外进行表达.

AIM: To construct the eukaryotic expression vector of human bFGF gene and to observe its expression in vitro.METHODS: Total RNA from bone marrow stromal cell (BMSC) was used to acquire human bFGF cDNA by RT-PCR.After the sequence was confirmed by sequencing,the eukaryotic expression vector was constructed,which was confirmed by restrictive enzymes (BamHI/EcoRI) digestion analysis,PCR and DNA sequencing.Similar nucleotide sequences encoding bFGF of pVAX1-bFGF were retrieved by Blast means in NCBI GenBank.The pVAX1-bFGF was transfected into BMSC by lipofectamine and the expressed product was detected by indirect immunofluorescence.RESULTS: Restrictive enzymes digestion analysis and sequencing results revealed that the recombinant expression vector pVAX1-bFGF was constructed successfully.The indirect immunofluorescence result showed green fluorescence on the membrane of the transfected cells.The sequence of encoding bFGF of pVAX1-bFGF was exactly the same as the reported sequence of encoding human bFGF in GenBank.CONCLUSION: The recombinant expression vector pVAX1-bFGF is successfully constructed and the constructed eukaryotic expression vector of pVAX1-bFGF can be expressed in vitro.