The Cloning Of Bovine Neutrophil β-Defensin-5 Gene And Expression In The Pichia Pastoris

Defensins are an ancient molecular, they act as an important role in host defense of innate immune system. They can activation the first defensin barrier of host and startup innate and achieved immuoreaction in bacteria, mildew, plant, invertebrate and vertebrate. Beta-Defensin genes can be induced to express in galactophore of mastitis cows. This phenomenon indicates that they possibly participate in part defense of galactophore. It has also found that mastitis can increase the expression quantity of BNBD5 for about 13 fold. Our lab has used suppression subtractive hybridization differentially expressed cDNA library of peripheral blood leukocyte were constructed and primarily confirmβ-defensin gene as one of the candidate genes. Therefore, bovine defensins can be candidate genes of mastitis for further study. Bovineβ-defensin 5(BNBD5) was first seperated from bovine neutrophile, its pre-peptide encodes 64 amino acids, molecular weight is 7228Da, can restrain the vivid of E. coli ML35, it is a secredted protein.This experiment using the blood of Hostein cow which trouble with mastitis. After extracted RNA from leucocytes of blood, I designed primers according to the cDNA sequence of bovine defensins in GenBank. Then Using RT-PCR technology to amplify the cDNA sequence of BNBD5, and cloned to pMD19-T vector. The plasmids which have been sequenced proved to be right named BNBD5/pMD19-T. Then designed primers added EcoRⅠand XbaⅠrestricted enzyme sites at the end of 5'. The fragment of mature BNBD5 gene was amplified from recombinant plasmid BNBD5/pMD19-T, the size is about 195bp. Then insert into the EcoRⅠand XbaⅠsites of Pichia pastoris expression vector pPICZαA, which carries anα-factor secretion signal sequence for excreting recombinant protein into the culture and a Zeocin marker for antibiotic selection. The recombinant pPICZαA/BNBD5 plasmid was linearized by restriction enzyme SacⅠ, then transformed into Pichia pastoris GS 115 by electroporation and chemical transformation, integrating into the downstream of the highly efficient AOX promoter in the Pichia pastoris chromosome. Under the selective pressure of high concentration of antibiotic Zeocin, high copies transformants were acquired. The transformants were identified by PCR using the bacteriam liquid as the templates. The positive transformed strains were cultured and induced by addition of 1％methanol every 24h. Expression product with molecular weight approximately 7.8kD was displayed on the Tricine-SDS-PAGE, showing that the BNBD5 gene was expressed in the Pichia pastoris.In summary, the above data demonstrated that the coden region of bovineβ-defensin 5 gene was successfully amplified and constructed the Pichia pastoris expression vector and first time using this expression expressed the BNBD5 protein. This research is the foundation of further researching the antimicrobial vivid, purifying recombined protein and preparing antibody.