Studies On The Cloning, Expression, Bioactivities And Application Of Human Augmenter Of Liver Regeneration Gene

Augmenter of liver regeneration (ALR) is a special kind of factor for promoting liver cell to split. ALR play an important role in the repairing process after liver damage. Liver disease is one of the key diseases, which endanger human health. The number of the patients of hepatitis, hepatocirrhosis and liver cancer is about 100 million, and the incidence of liver disease are increasing every year. According to the statistic of 1999, the incidence of liver disease is around 12%. So, in order to relive the suffering of the patients who have liver disease, it is very urgent to study a new kind medicine to cure liver disease. The scholars of our country have acquired obvious curative effect by using the mixture of fetus liver to cure every kinds of liver disease since 1987. From this point of view, the fetus liver must have the factors that stimulate the liver cell multiplication. From the liver of the weanling big rat Hagiya et al. separated one kind hot stability cell factor in 1994. Because it could promote liver cell multiplication, so it was named augmenter of liver regeneration (ALR).This dissertation uses the gene engineering method to clone, express and purifies the human ALR gene (hALR). It established a fundamental for further studying the hALR's bioactivities. We systemically studied the bioactivities of the hALR acquired by gene cloning method by establishing the big rat liver fibrosis model and small rat fat liver damage model. The work settle the fundamental for earlier using hALR in clinical to cure liver diseases. The results showed that:1. For the first time to extract the total RNA from fetus liver and use PT-PCR method to clone the cDNA of the whole reading frame of the hALR. The sequence analysis result showed that the recombine factor is consisted of 378 basic pair. However, two basic pair are mutation, which don't cause the amino acid mutation.2. For the first time to recombine ALR procaryotic expression vector, and get the pET28a-hALR recombined expressing plasmid which was tested by sequence analysis.3. For the first time to express pET28a-hALR gene induced by IPTG grads. The different concentrations of IPTG induce showed that the expressing quantity is highest when the IPTG concentration is 1M., For the first time to express pET28a-hALR gene induced by different times, the result showed that the expressing quantity is highest during the 3 to 4 hours of the induce.4. For the first time to analysis the pET28a-hALR gene expressing location. The protein of procaryotic expression exists both in the supernatant fluid and the sediment. It was more in the sediment than the supernatant fluid. That means the protein has a higher concentration in the E.Coli cell, and exists in the inclusion body.5 By Western-blot method to examine the expressing protein, and showed that there are a black bolting band around 20KD, which is interesting protein that we want.6 We use the Ni ion exchanging resin to purifying the protein. The SDS-PAGE of the purified protein has only one band, which showed that the protein was purified.7 For the first time to study the rhALR's bioactivities to liver fibrosis by establishing a human blood albumin immuno-damaging big rat liver fibrosis. The result showed that rhALR has an obvious inhibitory effect to the fibrosis tissue of the immuno-damaging liver. Applying rhALR during the immune liver fibrosis can significant lower the level of ALT, AST in serum and make the Alb level increase. It protects the liver cells form the immuno-damaging caused by human blood albumin.8. For the first time to study the rhALR's bioactivities to fat liver damaging by establishing small rat fat liver damaging model caused by ESAA. The result showed that the rhALR can inhibitor the ALT, AST activities to increase during fat liver, meantime lower the TG, TCh content The rhALR has curative act to the fat liver caused by ESAA9. The hALR can be produced in big scale by gene engineering, in this way not only reduce the cost, but also overcome the shortcoming of...