Clone And Expression Of Several Mastitis-associated Genes And Their Expression In Leukocytes

Mastitis of dairy cows is a serious disease which is of great damage to dairy industry; however, there is no efficient method to deal with. Recently, it was studied at molecular level with the development of molecular biology, so did the mastitis-associated genes.Chinese Hosltein cow leukocytes were used to extract total RNA and the mRNA expression level ofβ-defensin 5, MIP-3, L-selectin, S100A9 and S100A12 in leukocytes of dairy cows with or without mastitis was detected using semi-quantitative RT-PCR technique. In addition, MIP-3 gene andβ-defensin 5 gene were cloned, and the percent identity was analyzed. Then, theβ-defensin 5 gene mature peptide fragment was amplified according the recombinant clone vector plasmid. The gene fragment amplified was inserted into prokaryotic expressive vector pET28a after digested with EcoR I and Not I to form recombinant expressive plasmid. Furthermore, the recombinant expressive plasmid was transformed into Escherichia coli BL21(DE3) and induced with IPTG. Then, the expressive product was detected by Tricine-SDS-PAGE.The current results showed that the gene mRNA expression levels ofβ-defensin 5, MIP-3, S100A9 and S100A12 increased significantly in mastitis cow; L-selectin was not but holding on a same level. Both genes were correctly cloned and identified by PCR, restriction enzyme digestion and sequencing. And the MIP-3 gene cloned was completely consistent with that on the GeneBank. The identity of MIP-3 gene in Homo sapiens, Pan troglodytes and Mcacaca mulatta was high, but low in Canis and lowest in Bos taurus. Theβ-defensin 5 gene shared a 82% identity with the BNBD5 gene reported on the GeneBank. Eight amino acid residues might be mutation, that were: the ninth amino acid residue(P→L), the tenth amino acid residue(Q→V), the fourteenth amino acid residue(W→I), the sixteenth amino acid residue(M→R), the eighteenth amino acid residue(V→F), the twenty-eighth amino acid residue(M→T), the thirty-fifth amino acid residue(F→L) and the fortieth amino acid residue(P→K). Theβ-defensin 5 gene identity of different Gaulus species was high; however, it was the lowest between Bos taurus and Gallus, but Homo sapiens and Rattus norvegicus were higher than them. The identity betweenβ-defensin 1,β-defensin 4,β-defensin 7,β-defensin 8,β-defensin 10, LAP and TAP was high, so did it betweenβ-defensin 122 andβ-defensin 122a.β-defensin 119 andβ-defensin 120 also shared the similar identity. However, the identity betweenβ-defensin 5 gene and any other defensins was lower. The recombinant bacterial could express the aimed protein which was identified by Tricine-SDS-PAGE. It was obtained to the biggest mount of soluvable protein which weighed 6.4kDa when the recombinant bacterial were induced with IPTG at the concentration of 0.5mM, and the inducing temperature was 37℃.