Expression Of The Defensin Genes Defcr4 And Mcp-1 In Cow Mammary Glands

Mammalian Defensins are 29-36 amino acid antibiotic and cytotoxic peptides highlyabundant in the cytoplasmic granules of polymorphonuclear They play an important rolein oxygenindependent microbicidal mechanisms. Defensins exert in vitro microbicidalactivity against many bacteria, fungi and some enveloped viruses. In thisexperiment,defensin gene Defcr4 and Mcp-1 were cloned by primers synthesis ,RT-PCRand PCR splicing method. The mammary gland-specific expression vectorpBC-neo-Defcr4 and pBC-neo-Mcp-1 were constructed by insert a screening tag neo anddefensin genes into the enzyme site Not1 and Xho1 in the pBC1 vector respectively. andthen the Defcr4 expressed successfully in cow mammary glands by inject directly. TheDefcr4 with satisfactory antibiotic activities was extracted and purificated from milk bytwo-step RP-HPLC, the LC-MS was also used to detect Defcr4.This study provides thebasis to get the established cell line and further study on the production of the transgenicanimal.According to the gene sequence of the Decr4 and bovine β-Casein signal peptide inGenebank, we designed and syntheised 17 pieces of specific primer, and then amplifiedthe Decr4 coding sequence with bovineβ-Casein signal peptide by PCR splicing method.Then connected it with pGEM-T vector and cloned the syntheised gene.After PCRanalysised and sequenced ,the results conformed to the expected.In order to expression the cDNA of rabbit macrophage cationic peptide-1(Mcp-1) ineukaryotic cell, total RNA were isolated from rabbit spleen cells and the cDNA encodingof the Mcp-1 was amplified by the reverse transcription PCR(RT-PCR). Then connectedit with pGEM-T vector and cloned.The cDNA was identified by the PCR,restrictionendoncleases and sequenced, it was found there was only one difference in nucleotideas...