Expression,Eptope Analyasis And Antibaody Preparation Of Human Alanineaminotransferase-â… 

Alanine aminotransferase(ALT),also named glutamate pyruvate transaminase, is a key enzyme for gluconeogenesis and amino acid metabolism . ALT is mainly distributed in tissue of the liver. An elevated serum ALT activity is regarded as evidence of liver damage, including hepatitis,nonalcoholic steatohepatosis,fatty liver,cirrhosis and drug hepatoxicity. Analysis of serum ALT activity levels is routinely used for detecting liver injury, a biomarker that has been in use for more than 50 years.The serum ALT is mainly detected by the method of it's activity in the practice of medicine. Although the method for enzymic activity detection is relatively simple and accurate during operation, but to rely on conventional biochemical testing equipment, can not meet the need for rapid detection of ALT on-site the source of blood donation. And the activity assay just reflects the total serum ALT activity, rather than the actual quality of ALT, test results will reflect more exactly to the liver state.Three forms of ALT have been identified, ALT₁,ALT₂andALT₂-2. Experimental study found that, ALT₁ and ALT₂ share a common catalytic activity, while ALT₂-2 was not found to have catalytic activity. ALT₁ was mainly in the liver, skeletal muscle and kidney tissue, subcellular localization in cytoplasm and endoplasmic reticulum; while ALT₂ was mainly in cardiac cells and skeletal muscle, no expression in liver and kidney tissue, subcellular in the mitochondria and endoplasmic reticulum, not exist in the cytoplasm.Therefore, we intend to establish a diagnosis method based on immunological detection techniques, to detect the actual content of serum ALT₁ and meet the need for blood screening of blood donation on the spot. In order to establish in vitro diagnostic method for ALT₁, we project to prepare the specific antibody of ALT₁.Objective:After acquireing the truncated ALT₁ protein by prokaryotic expression system , we immunized New Zealand white rabbits for the preparation of the specific ALT₁ polyclonal antibody.Simultaneously, we immuned BALB / c mices with the synthetic peptides,and prepared the specific ALT₁ monoclonal antibody. Made the foundation to the development of in vitro diagnostic test of ALT₁.Methods:1. We constructed expression vector of the truncated ALT₁ by the technique of molecular cloning using the pCold-TF vector.2. Expression vector with ALT₁ were transformated into E.coil BL21(DE3),then the recombinant proteins were induced by IPTG ,after beening analyzed the express pattern by SDS-PAGE, proteins were purified by Ni2+ affinity chromatography,then cut the TF tag-protein by HRV-3C protease,in the end ,we acquired the truncated ALT₁ protein by gel filtration.3. Considering results coming from different B cell epitope prediction softwares,two epitopes-MC15 and CK17, were got to prepare the ALT₁ monoclonal antibody.We Submitted the sequences to the company to synthesis peptides and coupled with the protein carrier KLH to increase the immunogenicity.4. ALT₁ polyclonal antibody was prepared after the rabbits being immunied with purified truncated protein.5. ALT₁ monoclonal antibody was prepared after BALB/c mices were immunized with the coupled peptides -MC15 and CK17.Results:1.Truncated ALT₁ expression plasmid was constructed successfully after the identification by PCR and restriction enzyme digestion,which appeared a gene fragment consistent with the expected size. Also confirmed the same by gene sequence analysis, without any frameshift mutations.2.Pure recombinant protein was acquire after being purified by Ni2+ affinity chromatography.And truncated ALT₁ protein was purified by gel filtration after being digested by HRV-3C prolease. 3. High specificity and titer ALT₁ polyclonal antibody was acquired after rabbits being immunized with purified truncated protein.4. Hybridoma cells were acquired after BALB/c mices being immunized with synthesis peptides. And ALT₁ monoclonal antibody was acquired after mices being injected with hybridoma cells though abdominal cavity.Also the McAb titer was detected up to 106 by ELISA. Certainly,the McAb could recognize the native ALT₁ in the serum.Conclusions:Pure truncated ALT₁ protein was acquired after being purified by Ni2+ affinity chromatography and gel filtration. High specificity and titer ALT₁ polyclonal antibody and monoclonal antibody were acquired after rabbits or BALB/c mices being immunized with purified truncated protein or coupled peptides,which made a foundation to the development of a rapid in vitro diagnostic test strip of ALT₁.