Hp450 Gene And Its Recombinant Retroviral Vector Plxsn-hp450 Of The Building And Identified

OBJECTIVE To provide a good basis for studying liver cancer gene therapy in vitro and in vivo, HP450 gene was amplified from normal liver by reverse transcriptase-polymerase chain reaction (RT-PCR), and recombinant vector pMD18-HP450 and pLXSN-HP450 was constructed using plasmid pMT-18 and reverse transcriptase vector pLXSN respectively.METHODS 1. The total RNA of normal liver tissue from mice was isolated using Trizol reagent. 2. The HP450 gene was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and then cloned into pMD18-T vector. The positive clone was obtained by screening using several methods, such as screening with X-gal and IPTG, PCR, restriction digestion and sequencing. Finally, the sequence was submitted to the NCBI blast search (http://www.ncbi.nlm.nih.gov/BLAST/). 3. To get recombinant vector pLXSN - HP450 gene, the HP450 gene was removed from the vector pMD18-HP450 by digested with restriction enzymes BamHI and EcoRI. At the same time vector pLXSN was also digested with restriction enzymes BamHI and EcoRI. And then HP450 gene was ligated with pLXSN at 16oC overnight. Finally, the positive clone was identified by PCR and enzyme digestion.RESULTS 1. The total RNA was not degraded and has higher quality. 2. The HP450 gene with molecular weight of 1461bp was amplified by RT-PCR. The HP450 gene was ligated with pMD18 and then transfered to competent cells DH5α. After screening, the positive clone was identified. This clone was amplified to one gene with molecular weight 1461 bp. In addition, one product with molecular weight 1461 bp was obtained when this clone was digested with restriction enzyme BamHI and EcoRI. The sequencing result showed that this gene was found to be aligned with known gene in the GenBank, it shared nearly 100% nucleotide homology with H1. 3. After being digested pMD18-HP450 and the vector pLXSN with enzyme BamHI and EcoRI separately, the gene HP450...