Cloning, Construction Of Eucaryote Expression Vector And Procaryotic Expression Of Human Augmenter Of Liver Regeneration Gene

Human augmenter of liver regeneration(hALR) is a novel cytokine which stimulates specifically hepatic cell proliferation and is able to rescue acute liver failure caused by hepatotoxin for example carbon tetrachloride and galactosan amine et al. The object of this study is hALR, focus on its molecular cloning, construction of eucaryote expression vector and procaryotic expression.The experiment extracted total RNA from 6 month-old fetal liver, and amplified hALR gene fragment by twin steps RT-PCR technique. Target gene was cloned into the procaryotic fusion expression vector PET28a(+), then subcloned into the eucaryote expression vector pcDNA3.1(+) after sequence analysis. Procaryotic expression vector recombinant was transformed into competent BL21, induced and expressed by IPTG revulsant, explored the best inducing time and the best concentration of revulsant. Expressed protein was analyzed to determine quality and quantity by Western-blot and Scion Image 4.02 Beta software.This experiment cloned successfully 431bp hALR gene fragment After sequencing and comparing with Genebank, it was the cloned gene had 3 base difference (36tiu 177th,312th) . The homology of nucleotide acid sequence between cloned hALR gene and published one was 99.4%. The construction of eucaryote expression vector was accomplished, and the prophase preparation for eucaryote expression of hALR gene was made. Western-blot analysis proved that fusion protein of molecular weight 18.7 KD was gained after procaryotic inducement and expression. It was discovered firstly that hALR also expressed a little when it was not induced. When ultima concentration of IPTG was 1.0mmol/L and induced for 3 hours, the protein expression quantity was the mostCandidate: Zhao Chunli Major: Basic Veterinary ScienceSupervisor: Prof. HaoYanhong Prof. LiQingzhang...