Gene Cloning, Expression And Activity Detection Of SPLUNC1 In Sheep

Mycoplasma pneumonia in sheep is chronic respiratory disease, which cause the sheep cough, gasp, progressive emaciation and pulmonary interstitial hyperplastic inflammation, is harmful to sheep production. SPLUNC1(short palate,lung and nasal epithelium clone 1)is a protein molecules, which expressed highly on respiratory epithelium and involved in the host innate immune activity, has antibacterial and anti-inflammatory function, and significantly inhibite the growth of mycoplasma pneumoniae.Object:By cloning of full-length SPLUNC1 c DNA sequence of Hu sheep and Bashibai sheep to analysis its bioinformation in this research. Through eukaryotic expression of Hu sheep and Bashibai sheep SPLUNC1 gene to study the inhibitory effect of recombinant SPLUNC1 protein on Mycoplasma ovipneumoniae, and verify its biological activity to lay the foundation for further study the biological function of sheep SPLUNC1 protein.Methods: 1) amplification SPLUNC1 c DNA full-length of Hu sheep and Bashibai sheep: According to SPLUNC1 gene sequence of bovine and goat reported in Gen Bank to design primers, using rapid amplification of c DNA ends to clone 5’end sequence and 3’end sequence of SPLUNC1 of Hu sheep and Bashibai sheep, sequencing and splice to get full-length sequence. 2) bioinformatic analysis of Hu sheep and Bashibai sheep SPLUNC1 c DNA: using bioinformatics software and online analytical tool to analysis nucleic acid of Hu sheep and Bashibai sheep SPLUNC1 c DNA obtained, and its encoded protein’s signal peptide, subcellular localization, secondary structure, three dimensions and phylogenetic tree. 3) cloning and expression of Hu sheep and Bashibai sheep SPLUNC1 gene:using PCR to amplify the open reading frame sequence of Hu sheep and Bashibai sheep SPLUNC1 gene, this sequence was linked with a p PIC9 K vector to get the recombinant p PIC9K-SPLUNC1 vector, then the recombinant vectors were transformered into competent cell P.pastoris GS115 and induced, optimized the induce conditions,analyzed by Western Blotting and SDS-PAGE. 4)the purification and biological activity test of the recombinant Hu sheep and Bashibai sheep SPLUNC1 protein: using Ni-NTA to purified the recombinant Hu sheep and Bashibai sheep SPLUNC1 protein, and using the purified protein to acting on Mycoplasma ovipneumoniae, test its biological activity by realtime fluorescent quantitative PCR.Results and conclusion: 1) SPLUNC1 full-length c DNA of Bashibai sheep is 1095 bp, Hu sheep is 1091 bp. 2) There has five nucleotide bases differences on Hu sheep and Bashibai sheep SPLUNC1 c DNA, but their open reading frame sequence are 768 bp, encoded amino acid are same. The SPLUNC1 protein molecular is 26.53 KD, and its theoretical isoelectric pointis is 5.07. SPLUNC1 protein contained signal peptides, and were located extracellularly.SPLUNC1 protein secondary structure are α-helics and random coil. SPLUNC1 protein was composed by beta pleated sheet and bucket structure domain, which formed by four parallel peptides and two alpha helix, respectively. The phylogenetic tree shows that the protein of Hu sheep was so close to Capra hircus, then close to Bos Taurus, and this result is consistent with zoological classification. 3) Successfully expressed Hu sheep and Bashibai sheep SPLUNC1 protein in P.pastoris GS115, the result of SDS-PAGE showed that the molecular weight Hu sheep and Bashibai sheep SPLUNC1 protein is 25.53 k D, and found that the protein expression level is the highest on the methanol induction 72 h, the Western Blotting detection vefied the expressed protein is target protein. 4) SDS-PAGE test the puried recombinant protein, the result showed that there has a single band and the molecular weight is 25.53 k D, it suggested that we successfully puried the target protein.The realtime fluorescent quantitative PCR showed that the recombinant Hu sheep and Bashibai sheep SPLUNC1 protein has significantly inhibition effect on mycoplasma pneumoniae, it suggested that Hu sheep and Bashibai sheep SPLUNC1 protein expressed has a good biological activity.