Preparation Of Antiserums And Expression Characterization Analysis Of Bovine A-FABP

Specific primers were designed according to Genbank sequences (Accession No: NM₁74314) to amplify cattle A-FABP gene by RT-PCR. Then the A-FABP PCR product was inserted into pET-30a vector and expressed in Escherichia coli. The fusion proteins were purified by Ni-NTA affinity chromatography. And then the antiserum against A-FABP was produced by immunizing rabbit with the purified fusion proteins and high titer antiserum against A-FABP was obtained. Finally, expression characteristics of A-FABP were analyzed on mRNA level by Real-Time PCR, and on protein level by Western Blot. In our study, the sequence of cattle A-FABP was also analyzed by DNAMAN, NCBI, and some other software and inline tools in biological information. Our current study will provide lots of contribution to gene function future study of cattle A-FABP. The main results are as follows:1. Antiserums against cattle A-FABP was successfully prepared.The full-length CDS of cattle A-FABP was amplified by RT-PCR, and the prokaryotic expression plasmids of A-FABP were constructed. A-FABP fusion protein was induced and purified; high titer (1:3200) antiserum against A-FABP was prepared.2. Tissue expression of cattle A-FABP was characterized.The expression characteristics of A-FABP on mRNA level were analyzed by Real Time PCR. The results showed that A-FABP was only highly expressed in fat and was nearly not expressed in heart; liver; spleen; lung; kidney; small intestine and muscle. The expression characteristics of A-FABP on protein level were also analyzed by Western blot. The results showed that A-FABP was expressed in fat and was not expressed in heart; liver; spleen; lung; kidney; small intestine and muscle.3. The sequence of cattle A-FABP was analyzed in biological information.The analysis of amino acid sequence indicated that cattle A-FABP gene has 15 restriction enzyme cutting sites, and codes 132 amino acids, molecular weight 14.7kDa, mainly elements of its secondary structure areα–helix, random coil and extended strand, including 6 protein kinase C phosphorylation site, 1 casein kinase phosphorylation site, 3 N-myristoylation site and 1 cytosolic fatty-acid binding proteins signature, and also including 4 Ser, 6 Thr and 1 Tyr, which could be protein kinase phosphorylation site. Cattle A-FABP includes lipoid-calcium bind domain, but not including signal peptide, transmembrane domain, and disulfide bond. Homologous comparison with some animals indicated that cattle A-FABP shared 84.09%, 85.61%, 71.97%, 88.33%, 81.82% similarity in nucleic acid sequence with human, chicken, mouse, rats, and swine. Bioinformatics analysis indicated that A-FABP gene has highly conservatism.