藏猪β-防御素2基因克隆及原核表达载体的构建

发布时间：2018-05-12 12:09

本文选题：藏猪 + β-防御素　；参考：《江苏农业学报》2017年04期

【摘要】：为了构建藏猪β-防御素2(pBD-2)成熟肽原核表达载体,从藏猪肝组织中提取总RNA,通过RT-PCR获取pBD-2目的基因,并与pMD19-T载体连接构建重组质粒,命名为pMD19T-pBD-2;以pMD19T-pBD-2质粒为模板,克隆pBD-2成熟肽c DNA序列并构建成熟肽p ET-32a-pBD-2表达质粒。结果显示,藏猪pBD-2基因全长为298bp,与野猪和家猪的pBD-2基因同源性分别达100.0%和98.9%;藏猪pBD-2基因具有1个完整的开放阅读框,编码69个氨基酸组成的多肽,包含21个氨基酸残基的信号肽、11个氨基酸残基的前导肽和37个氨基酸残基的成熟肽;编码的pBD-2成熟肽稳定性较好,分子量4 085.78,等电点8.89,具有6个半胱氨酸(Cys)残基,7个带正电荷和2个带负电荷的氨基酸残基,为亲脂性和亲水性多肽;从pMD19T-pBD-2质粒中亚克隆得到长度为114 bp藏猪pBD-2成熟肽c DNA序列,菌落PCR、酶切和测序鉴定证明成功构建了成熟肽pET-32a-pBD-2原核表达质粒。
[Abstract]:In order to construct the prokaryotic expression vector of 尾 -defensin 2pBD-2) mature peptide, total RNAs were extracted from Tibetan pig liver tissue, and the target gene of pBD-2 was obtained by RT-PCR. The recombinant plasmid was named pMD19T-pBD-2, which was named as pMD19T-pBD-2. The recombinant plasmid was named pMD19T-pBD-2. The sequence of pBD-2 mature peptide c DNA was cloned and the expression plasmid of mature peptide p ET-32a-pBD-2 was constructed. The results showed that the total length of pBD-2 gene of Tibetan pig was 298bp. the homology of pBD-2 gene with wild pig and domestic pig was 100.0% and 98.9.The pBD-2 gene of Tibetan pig had a complete open reading frame and encoded 69 amino acid polypeptides. The signal peptide containing 21 amino acid residues, the leading peptide of 11 amino acid residues and the mature peptide of 37 amino acid residues have good stability. The molecular weight is 4 085.78, the isoelectric point is 8. 89, with 6 cysteine cysts, 7 positively charged and 2 negatively charged amino acid residues, which are lipophilic and hydrophilic polypeptides. The prokaryotic expression plasmid of mature peptide pET-32a-pBD-2 was successfully constructed by colony PCR, restriction endonuclease digestion and sequencing.
【作者单位】：四川农业大学动物医学院动物微生态研究中心;成都农业科技职业学院;绵阳泰科生物技术有限公司;
【基金】：四川省高等学校科技创新团队资助项目(KM406183.1) 四川农业大学学科双支计划项目(03570434)
【分类号】：S828

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