结肠癌肝转移灶人PRL-3基因的扩增与克隆

Amplification and cloning of phosphatase of regenerating liver-3 from the humans

目的从人结肠癌肝转移病灶标本中提取染色体RNA,聚合酶链反应(PCR)扩增及克隆人PRL-3基因。方法收集人结肠癌肝转移标本,提取总RNA,逆转录为cDNA,PCR扩增 PRL-3序列,A-T克隆于pMD-18T载体中,转化大肠杆菌JM109株,提取质粒,对重组质粒进行酶切与测序鉴定。结果通过酶切、PCR与测序3种方法证实所获得的基因片断为PRL-3基因。结论克隆到序列正确的人源性PRL-3基因,为实现PRL-3基因的高效表达及制备相应抗体奠定了基础。

Objective To amplify and clone the coding sequence of phosphatase of regenerating liver-3 （PRL-3） from the human liver metastasis samples of colorectal cancer. Methods Extract genomic RNA from human liver metastasis samples, reverse transcript to cDNA , and amplify the coding sequence of PRL-3 By using PCR technique,then the gene was linked to pMD-18T vector and transformed to E. coli JMI09. The recombinant plasmid was identified by Barn H I and Hind Ⅲ digestion and by sequencing. Results It turns out that the cloned sequence is PRL-3 gene by Barn H Ⅰ and Hind Ⅲ diges- tion,PCR technique and sequencing. Conclusion The correct PRL-3 gene from the humans is successfully amplified,and it provides the basic researches for the high level expression of its coding protein and preparation of its antibody.