Study On Gene Clonning, Expression In E.coli And Elementary Effect Of Human Augmenter Of Liver Regeneration

The liver is the organism which has formidable ability of regeneration. At present, the known liver regeneration facters like liver growth factor (HGF), transformation growthfactor -α(TGF-α), epidermis growth factor (EGF) all have difficulty to explain the regulation of the organ specificity liver regeneration. So seeking the new liver regeneration regulation factor is a research hot spot in this field.In 1994, Hagiya cloned a new kind of the liver cell multiplication factor of the big rat for the first time. It is named augmenter of liver regeneration(ALR). Some people also called it hepatopoietin(HPO). ALR performs the vital role in the liver damage repair process.The first, people thought ALR has the liver specificity, but the expression of ALR mRNA out of the liver gainsaid this. Francavilla and his partner found that except liver lots of the rat's ALR was in the spermary but fewer in the heart, brain, spleen, lung, skeletal muscle and kidney.The 1.2 kb length of the big rat ALR DNA sequence is mainly composed by three parts: 299bp 5'-untranslated region, 375bp coding region and 550bp 3'- untranslated region. The human ALR cDNA is 1.5kb length, slightly larger than the big rat. Both of them haVE 87% homology on cDNA coding region. ALR has no relation with other liver cell growth factors on gene structure, however, its coding area is 50% homological with the nuclear gene of yeast fungus ERV1. ERV1 is a part of mitochondrial respiratory chain, and plays an essential role in the process of cell regulation. Through the sequence analysis on human 16th chromosomal and scERV1cDNA, the human homological gene sequence has been found near ploycystic renal site. ALR gene is likely alike with ERV1. It is an important growth regulation gene.The typical signal peptide sequence of eucaryon emiocytosis protein hasn't been discovered through the DNA and cDNA study in different ALR gene group. Therefore, there still exists problem to solve in the process of generation, secretion, and vivo transportation. Recent studies indicate ALR-coded gene is not so short. The previous gene sequence just codes the ALR mature protein, not containing signal peptide sequence. One of the most remarkable features is the mechanism of cutting and processing after transcription. Because of this very mechanism, it forms a larger mRNA which provides another amino-acid residue sequence at the amino terminal of the new ALR protein.ALR is a peptide regulatory factor. Because of its particular cutting mechanism, ALR exists in vivo in two format: one consists of 204 amino acids (ALR204), located in chondriosome, participating in oxidative phosphorylation respiratory function regulation. Another consists of 125 amino acids (ALR125), mainly taking part in liver adjustment process. The relative molecular weight of ALR is 15kd, and may exist homodimer structure in vivo. It has characteristics such as thermal stability, immunogenicity, neuramidinase-resistance and reductant function, and can be destroyed through trypsinization. This factor maintains stable nature under PH 4.5-7.5, without organ and species specificity and mainly consists of acidic amino acid.ALR exert its function by combining specific high affinity acceptor on liver cell membrane. Receptor protein's molecular weight is 75kda, and it is the key molecular mediate ALR biologic activity.The research indicates ALR performs remarkable therapeutical effect to both of acute hepatic injury and chronic hepatic injury. ALR can reduce ALT, AST, LDH level of hepatic fibrosis rat significantly, and still has some subsidiary effect on diabetes.Up to present, the research on ALR is only limited on the mechanism of action abroad, and achieved no remarkable progress. Domestically, Chinese People's Liberation Army Academy of Military Medical Sciences, Virus Hepatitis Institute of Chongqing Medical University all carried preliminary study on ALR expression, purification, and pharmacodynamic action. The former protokaryonly constructed, expressed, purified rhALR and completed Hepatic injury recovery test, while the latter made ALR constructed and expressed in Pichia pastoris. All these research achieved considerable progress but some obstacles still stay aheadThe main papers for the following aspects : 1) From the use of codon, adjustment G+C% content and distribution, the number of mRNA stability perspective on human augmenter of liver regeneration(ALR) to make an entirely new design, and synthetic human ALR genes.2) New synthetic gene was constructed into different pRSETB vector,pBV220 vector,pET20b(+) vector, and then transformed to engineering bacteria, expressed the recombinant hALR125 protein. To be the product of analysis by more pBV220 vector for the best expression of the vector, reflecting induce simple methods to induce expression of the largest, low-cost, easy to operate and other advantages.3) hALR125 protein sample separation and purity study. The hALR125 nonfusion protein was expressed and purified through ion exchange and Sephacryl S-100 purfication steps, available for more than 90% purity protein expression. We set up a laboratory-scale purification platform.4) In addition, the recombinant hALR125 was constructed into pRSETB (his) vector and purfied for the immunization of female rabbit to get hALR125 multi-clone antibodies.5) In pharmacodynamics test, we created mouse CCl4 acute hepatic injury model, and inject them using rhALR125 through vena caudalis according to each group's dose. We estimate rhALR125's effect in liver regeneration process through glutamate-pyruvate transaminase detection and liver pathological section observation. The result shows rhALR125-injected mouse sees remarkable reduction of both of glutamate-pyruvate transaminase and glutamic-oxal(o)acetic transaminase. pathological section also shows obvious protection effect, which illustrates rhALR125 does play an important effect on hepatic injury recovery.