正常人肝细胞cDNA文库的构建及应用分析

Construction and Identification of a cDNA Library from Human Liver Cell

目的利用Gubler-Hoffman法构建了正常人肝细胞的cDNA文库以筛选肝细胞内部与乙肝病毒感染相关的基因。方法首先采用TRIzol法提取正常人肝细胞总RNA,纯化mRNA。逆转录合成单链cDNA,然后合成双链cDNA。用Spin Column回收0.4kb以上片段,然后与Vector pAP3neo进行连接,利用电刺激转化法导入E.coliDH10B,利用PCR法检测文库的重组效率。结果扩增后的文库重组率为93.3%。结论已经成功地构建了正常人肝组织的cDNA文库,该文库可用于筛选与乙肝相关的基因及用于基因芯片的制作。

Objective To screen genes associated with HBV,a cDNA library of normal human liver cell was constructed with the methode of Gubler-Hoffman, Methods Total RNA was extracted with TRIzol and mRNA was purified.mRNA was reversed to first-strand cDNA which was amplified to double-strand cDNA.The cDNA of length longer than 0.4kb were collected and ligated to pAP3neo vector.The plasmids were transformated into E.coli DH10B by electroporation,the quality of the cDNA library was test by PCR, Results The percentage of recombinants were 93.3%. Conclusions A high quality cDNA library from human liver tissue,which would be helpful to further detecting target genes associated with HBV and preparing gene chips etc,was constructed successfully.