【摘要】 为研发牛β-防御素生物制剂,防治奶牛乳腺炎。根据已发表的牛β-防御素基因序列设计一对引物。收集患乳腺炎奶牛乳静脉血液中性粒细胞,用试剂盒提取总RNA。经RT-PCR扩增牛β-防御素cDNA片段,回收纯化PCR产物,连接pMD18-T载体,转化感受态细胞JM109。在含X-Gal、Amp及IPTG的LB平板上筛选阳性克隆,经菌落PCR及双酶切鉴定后,对目的片段进行测序。结果表明RT-PCR扩增出的cDNA片段碱基数为189bp,含有一个完整的ORF,能编码63个氨基酸的多肽,多肽中含6个保守半胱氨酸残基。用Blast程序进行检索比较,表明扩增序列与GenBank中发表的BNBD-7编码区序列96.3%相同。结果成功克隆出奶牛β-防御素家族的成员BNBD-7基因,为重组牛β-防御素的开发奠定了基础。更多还原

【Abstract】 A specific pair of primers were designed and synthesized according to the sequence of bovine β-Defensins gene published in GenBank.Total cellular RNA was purified from the bovine neutrophil cells with the total RNA isolation kit.Then the bovine β-defensin cDNA fragment was amplified by reverse transcription-polymerase chain reaction(RT-PCR).After gel purification,the RT-PCR products were inserted into the pMD18-T cloning vector.Recombinant pMD18-T BNBD-7 vector was transduced into competent cell JM109.Clones containing the DNA fragment of insert were screened on the LB agar plate with X-Gal、Amp and IPTG.Restriction enzyme digests and PCR were performed to identify the clone containing the DNA fragment of interest,then the cDNA fragment of interest was sequenced.The result showed that the obtained 189 bp DNA fragment is identical to the BNBD-7 sequence registered in the GenBank.The cDNA fragment code 63 amino acid residues,which contained the consensus sequence of six invariantly spaced cysteine residues.Compared to bovine defensin,it shared the highest identity of 96.3% with the BNBD-7.The results show that the cloning and sequencing of bovine β-Defensins gene is successful.更多还原