Preparation Of Antibodies Against Human Alanine Transaminase â…  And Study Its Bionomics

Alanine transaminase,also known as glutamate pyruvate transaminase,is a pyridoxal enzyme catalyzing reversible transamination between alanine andα-oxoglutarate to form pyruvate and glutamate. By mediating the conversion of these four major intermediate metabolites,ALT has an important role in cellular nitrogen metabolism and liver gluconeogenesis. It is well known that ALT is widely used as a surrogate marker for liver function. Serum ALT activity is significantly elevated during liver damage caused by drug, toxicity,infection,alcoholand steatosis,for when liver damaged, a large amount of aminotransferase would be released into serum.At present ,the principle of clinical method to detect ALT in serum was on the basis of ALT catalyze L-alanine andα-oxoglutarate to form L-pyruvate and L-glutamate. by then,coupled an indicative reaction to detect and calculate ALT catalytic active concentration. It is well known that the enzyme active concentration detection methods are easily interfered by substrate concentration, Apoenzyme inhibitor, ionic strength,pH and temperature. Furthermore,the enzyme active concentration detection methods can not distinct different isoenzyme. So, It is stringent to establish specific immunologic methods to efficacious detect enzyme mass and distinct different isoenzymes to objective reflect liver function.Objective:To construct a prokaryotic expression vector ATT1,to purify and identify ALT1 protein produced by the expression system and to prepare its polyclonal antibody after rabbits were immunized with the fusion protein ,which provide a basis of establishment a clinical immunoassays for human serum ALT1.Furthermore Balb/c mice were immunized with the recombinant protein,and the monoclonal antibody was produced,which could provide a basis of establish an immunoassay methods and explore deeply its biological roles.Methods:1. Total RNA was isolated from HepG2 cell line, and human ALT1 gene was amplified with RT-PCR.The code fragment was cloned into prokaryotic expression vector.2. The vectors were transformed into E.coil BL21 (DE3) in which ALT1 expression was induced, the protein was identified by Western Blot, then purified through affinity chromatography.3. ALT1 polyclonal antibody was prepared after rabbits were immunized with purified ALT1 fusion protein.4. ALT1 monoclonal antibody was prepared after Balb/c mice were immunized with purified ALT1 fusion protein.Results:1. RT-PCR amplified the fragment of the gene which is identical with expected fragment. The clone is successful proved by double enzyme digestion and sequencing of the recombinant plasmid which consists of ALT1 gene.2. ALT1 fusion protein was produced by recombinant expression purification, which presented in the inclusion body of pET32a (+)/ALT1 and slightly presented as soluble form in the supernatant of pGEX-4T-2/ALT1.The fusion protein can all be purified by affinity chromatography.3. ALT1 polyclonal antibody can be produced by immunized rabbits with purified ALT1 fusion protein.4. One hybridoma cell lines were achieved successfully with hybridoma technique,which could stably secret specific McAb against ALT1.After one of the hybridoma cell line was injected into mice abdominal cavity,the ascites abundant in McAb was obtained.Conclusions:ALT1 fusion protein is obtained by the method of genetic recombination technique. ALT1 polyclonal antibody and monoclonal antibody against ALT1 is prepared,which provide a basis of establish an ALT1 immunoassay methods and explore deeply its biological roles.