Cloning,Expression And Activity Analysis Of Two CDNAs Encoding Lipoprotein Lipase Of Cyprinus Carpio

Fat is an important energy storage substance in the body,and its decomposition product fatty acid is an important structural material and active substance.Lipoprotein lipase can break down fat,and it plays an important role in the regulation of lipid metabolism and lipid metabolism.There are two kinds of lipoprotein lipase(LPL1,LPLZ)in fish.At present,the research on the structure and function of comnon carp LPL has not been reported,so this paper selected carp as the research object,the expression and function of LPL gene in common carp were clarified.Using the method of RT?PCR to separate the LPL1 and LPL2 encoding cDNA full length sequence,The cloning results showed that the open reading frame of LPL1 and LPL2 were 1524bp and 1503bp,encoding 507 and 500 amino acids respectively,and the amino acid similarity was 45.51%.The analysis of functional sites of amino acids about LPL1 and LPL2 showed that the N-glycosylation sites were 85N,404N and 400N,the heparin binding domain were respectively 321R-323N and 319R-321N,the catalytic active sites respectively were 174S,198D,283H and 172S,196D,281H,and the dimer fomation conserved hydrophobic residues sites were 218A,230G,237G and 216A,228G,235G.Phylogenetic analysis showed that the genetic distances between LPL1 and LPL2 of common carp and the different classes of LPL2 and LPL1 are basically consistent with the classification status.The phylogenetic tree showed a good level of fish in different families and objective system.The expression of LPL2 and LPL1 in different tissues of common carp was detected by real-time fluorescence quantitative PCR The results showed that LPL1 and LPL2 were expressed in different tissues,and the expression of LPL1 and LPL2 in the liver was the highest,followed by heart,fat,muscle,brain,kidney,and in the foregut was the lowest.In all tissues the expression of LPL1 was higher than that of LPL2.The content of LPL in heart,fat,muscle,liver and other tissues of common carp was determined by the method of enzyme-linked immunosorbent assay and BCA total protein.It vas found that the results was consistent with the mRNA expression levels of LPL.The recombinant mature peptides of LPL1 and LPL2 were successfully expressed and purified in E.coli,The results of the analysis of the method of p-nitrophenol showed that the optimum temperature for LPL1 and LPL2 enzyme activity was 35?,and the optimum pH was 8.Under the optimum temperature of 35?and pH8 conditions,the enzyme activity of LPL1 and LPL2 were 22.69U/g and 17.4U/g respectively.It was clear that the difference between the two sequences resulted in different protease activities.