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中国农学通报

中国农学通报 ›› 2009, Vol. 25 ›› Issue (10): 6-10.

所属专题： 生物技术； 畜牧兽医

• 畜牧 动物医学 蚕 蜂 • 上一篇    下一篇

逆转录病毒介导的天蚕抗菌肽B在奶牛乳腺中的表达

王铁东,逄大欣,欧阳红生   

  • 收稿日期:2009-02-05 修回日期:2009-03-18 出版日期:2009-05-20 发布日期:2009-05-20

Retrovirus-mediated Expression of Antibacterial Peptide Cecropin B in Mammary Gland of Bovine

Wang Tiedong, Pang Daxin, Ouyang Hongsheng   

  • Received:2009-02-05 Revised:2009-03-18 Online:2009-05-20 Published:2009-05-20

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摘要： 摘要：为了研究天蚕抗菌肽在奶牛乳腺组织较长期表达的可行性及对天蚕抗菌肽对大肠杆菌的抑制作用，将天蚕抗菌肽B(cecropin B,CB)的基因序列,以哺乳动物偏爱的密码子优化后，设计合成了四条相互重叠的DNA片段，通过重叠延伸PCR法合成了天蚕抗菌肽B的基因，亚克隆至T载体，测序正确后构建逆转录病毒载体pLNCX-bCP-cecB，经过包装细胞的包装，获得含有天蚕抗菌肽B表达框的逆转录病毒，注射入奶牛乳腺组织,通过琼脂板孔穴扩散法检测基因的表达及活性；实验结果表明，克隆的抗菌肽基因在乳腺中获得了表达，表达产物对大肠杆菌具有抑制作用，抑制效果可以持续至13天以上。

关键词: 土壤含水量, 土壤含水量, 蔬菜产量, 水分渗漏

Abstract: Abstract: We synthesized the code-optimized gene of Cecropin B according to it’s natural sequence by overlap extension PCR, The gene was then cloned into the vector pMD-18T and was confirmed by DNA sequence. In order to get the whole mammary specific expression cassette, the gene of Cecropin B was cloned into the mammary specific expression vector pIRES-Bcp-neo which contain the goat β-casein gene 5'flanking regulation sequence, The whole mammary specific expression cassette was cloned into the retrovirus vector pLNCX, so the retrovirus vector pLNCX-bCP-cecB was constructed, This vector was transfected into a packaging cell line PA317, RNA from the vector is packaged into infectious, replication-incompetent retroviral particles which was then injected directly into the lactating mammary gland of bovines. The expression and bactericidal activity was detected using inhibition zone assay for the bacteria: E.coli K12D31, Antibacterial Peptide Cecropin B expressed in milk possesses bactericidal activity which can last at least thirteen days.