Studies On The Expression Level Of γPig Liver Esterase With Adult Pigs And Piglets

Liver is the main organs of animals (or humans) which remove the drugs, It expresses a variety of drug metabolizing enzymes, The y-isoenzyme of pig liver esterase is one of the most important and common Phase I drug metabolizing enzymes in liver. It could prompte the biotransformation of drugs which contains esters, amines and sulfur esters and activate the prodrugs.It also included the detoxification and metabolism of a variety of environmental toxins and carcinogens; the transportion and metabolism of lipid, transmembrane signal transduction and maintaining the integrity of the biofilm.Drugs in combined application is the most common way of current clinical medication, regardlessly, two or more kinds of drugs were used simultaneously or successively, there may exist the possible changes of drug therapy effect; the dose of the drugs in different day-old, different varieties of pigs was different in clinical medication; And these function is mostly mediated by the drug metabolizing enzymes. So, Establishment of y-PLE expression profile will provide a theoretical basis for for the problems existing in the clinical application.This research focused on developing and evaluating a method for the expression level of y-isoenzyme of pig liver esterase with different days, which was based on methods of Western blot, Real-time PCR techniques. The results were summarized as follows:1. The amplification of y-isoenzyme of pig liver esterase fragment, and the construction of recombinant plasmidThe mRNA of porcine liver was extracted, the porcine y-PLE gene was amplified by RT-PCR and PCR, y-PLE gene was about 1686 bp, then the gene was linked into the vector of pET-32a(+) after the digestion and recycling. The recombinant plasmids were identified by PCR, restriction-enzyme analysis and sequencing. The sequence showed that porcine y-PLE gene has the homology 99% with X63323 after BLAST. It has successfully established the recombinant expression plasmid in this research.2. The expression of y-isoenzyme of pig liver esterase fragment, purification and activity detectionRecombinant plasmid pET32a-yPLE was transformed into E. Coli BL21(DE3). Then finding out the optimization of expression conditions. The result shows that the recombinant protein was expressed at 37℃for 5 h. The molecular weight of the recombinant protein was about 75 KDa. The result of Western blot demonstrates that the recombinant protein has immunologic reactivity. The purity of recombinant protein was analyzed and purified from inclusion body. Finally the expression amount ofγ-PLE is about 2.01 mg/mL. It will provide a basis for the preparation of rabbit anti-γPLE polyclonal antibody. The result of activity detection demonstrates that the recombinant protein has the esterase activity.3. The preparation and purification of rabbit anti-γPLE polyclonal antibodyRabbits were immunized with the recombinant protein of pET32a-γPLE, several times later, had successfully prepared the hyperimmune serum of rabbit anti-γPLE. Then, the hyperimmune serum was crude extracted by saturated ammonium sulfate and purified by G-200, We had finally prepared the purified polyclonal antibody of rabbit anti-γPLE. The result of Western blotting demonstrates that the polyclonal antibody has immunologic reactivity. It will provide a basis for the study on the expression profile research ofγ-PLE.4. The study on the expression profile ofγ-PLEProteins were extracted from fresh tissue of piglets and adult pig liver.β-actin was probed and used as a measure for protein loading-Internal reference protein. The prepared and purified polyclonal antibody was used as a first antibody for the Western blot. The experimental result shows that the expression ofγ-PLE was higher in the adult pig; Furthermore, The total RNA was extracted from fresh tissue of piglets and adult pig liver. It was reverse transcriptased into cDNA. The gene of GAPDH was used as the internal reference gene. Compared with the piglets, The result of Real-time PCR demonstrates that the relative expression level ofγ-PLE in adult pig was more than 31 times. The difference was significant. In this research, The expression profile was confirmed from the levels of protein and mRNA.