Gene Cloning And Prokaryotic Expression Of Liver-type Fatty Acid-binding Protein From Grass Carps, Ctenopharyngodon Idellus

Liver-type Fatty acid-binding protein(L-FABP) as a member of Fatty acid-binding protein family is a key protein involved in the metabolism of long-chain fatty acids.It has a high affinity with long-chain fatty acids and help long-chain fatty acids transfer to the cell membrane which has sites of fatty acids oxidation and synthesis.In the study,L-FABP cDNA was cloned from grass carp (Ctenopharyngodon idellus) applying RACE(rapid amplification of cDNA ends)-PCR.The full-length cDNA of Liver type fatty acid-binding protein(L-FABP) was cloned from liver of grass carp.It consisted of a sequence of 487 bp comprising a 55 bp 5' untranslated region(UTR),a 51 bp 3'-UTR and a 381 bp open reading frame(ORF) encoding an peptide,consisting of 126 amino acids.Grass carp L-FABP mRNA mainly expressed in the liver,spleen,ileum,less in foregut,head kidney and body kidney.In the mid-intestine,muscle and gill there was nearly no expression by Reverse transcriptase polymerase chain reaction.The primers coding for the open reading frames of L-FABP were designed, which contain two enzymes sites,the same as vector pET32 contains.The ORF of L-FABP with was cloned into vector pET-32a(+) after digested by enzymes,then constructed a prokaryotic expression vector pET-32a-L-FABP with a T7 promoter. Recombinant plasmid identified by sequencing,and then transformed into E.coli BL21(DE3) for expression,then induced by IPTG.The products of expression was detected by SDS-PAGE.The result showed that a 33 KD fusion protein was obtained, including 49.5%of total protein.The result provided foundation for further to research to the biological function of fatty acid-binding protein on fatty acid metabolism.