Cows S100A12 Gene Cloning, Prokaryotic Expression And Antibacterial Activity Analysis Of Expression Product

The expression of S100A12 gene in peripheral blood and milk is able to be directly induced by mastitis, which enhances the defense of the quarter suffered mastitis, indicating that S100A12 plays an important role in the innate immune system of bovine mammary gland. In this study, based on genes acquired by successfully established mastitis resistance related cDNA library and reversed Northern blotting hybridization difference analysis, S100A12 gene in peripheral blood of cow was molecularly cloned and sequence analysed. Subsequently, prokaryotic expression vector pCold TF-S100A12, whose expression was induced in Escherichia coli BL21(DE3), was successfully constructed. Meanwhile, antibacterial activity of the recombination protein against Escherichia coli and Staphylococcus aureus which were considered to be the main cause of bovine mastitis was evaluated.1. S100A12 gene in bovine peripheral blood was obtained by RT-PCR which was then connected into pMDTM 19-T Simple vector. The successful establishment of restructuring cloning plasmid pMD19-T-S100A12 was proved by the plasmid PCR, double enzyme cut and sequencing. Bioinformatics analysis results showed that size of the target amplicon was 291bp with an integrated 279bp open reading frame (ORF) which coded a mature peptide consisted of 92 amino acids and with the specific molecular structure of S100A12 gene, namely EF-hand structure in specific position. Furthermore, three protein kinase C phosphorylation site were predicted, which located in 19-21,44-46 and 70-73 amino-acid residue, respectively. Similarity of the target amplicon and the previously reported bovine S100A12 cDNA(Accession number:NM₁74651) was 100% which completely covered the coding region. Cladogram analytic result showed S100A12 gene of bovine had a nearer genetic relationship with human, swine and simian.2. The expression of S100A12 gene in mammary gland and epithelial tissue in genital system(ovary, cervix, body of uterus, uterine tube, vagina) of bovine were found by application of RT-PCR.3. pMD19-cloning plasmid was cut by restriction enzyme BamHI and Hind III and production of which was connected with prokaryotic expression vector pCold TF. The results of plasmid PCR, double enzyme cut and sequencing showed that the establishment of recombinate prokaryotic expression plasmid pCold TF-S100A12 was successful. Subsequently, the recombinate plasmid pCold TF-S100A12 was transformed into BL21(DE3)strain with high effect expression. By induced culture, a 62kDa peptide was specifically expressed in the recombinate strain. Moreover, the most appropriate induced conditions (37℃,1.0 mmol/L IPTG, for 4h)were determined by optimization. Result of SDS-PAGE and western blotting indicated that the cloned S100A12 gene was expressed in form of fusion protein, accounting for 46.7% of total protein of the strain.4. Ni-TED affinity chromatography was employed to the purification of the recombinate expression protein which concentration was 0.5mg-mL-1, showed by the result of Bradford detection. Scraps of paper method determined the obvious antibacterial activity of recombinat expression protein against Escherichia coli. In contrast, it was negative against Staphylococcus aureus.The results and conclusion of this study provide with some foundation for the further understand of expression characteristic and function of bovine S100A12 gene and research for preparation of antibody and screening early diadynamic criteria, as well as development of recombinat peptide for precaution and therapy for bovine mastitis and breeding of diary cow with resistance to mastitis.