天蚕素 A - 蛙皮素杂合肽基因突变体的构建及其表达
onstruction of a Point-Directed Mutant of CA-MA Hybrid Peptide and Expression in E.coli
目的 用PCR定点突变法构建天蚕素 A(1～8)- 蛙皮素(1～12)杂合基因抗菌肽( CA-MA 杂合肽)突变体,并在大肠杆菌中融和表达。 方法 采用 PCR 体外定点突变技术,设计一对方向相反的引物,其中一个引物引入突变点,应用高保真的 Polybest DNA 多聚酶进行重组表达质粒 pGEX-4T-1-CA-MA 的 PCR 扩增,使 CA-MA 杂合肽第 16 位密码子由 AGT 变为 TGG ,将扩增片段自身连接，构建 CA-MA 杂合肽突变重组表达质粒,克隆入工程菌 E.coli BL21 中表达其突变肽 W16-CA-MA。 结果 DNA 测序结果表明在预期位点发生突变,突变体在大肠杆菌中高效表达。 结论 获得突变肽 W16-CA-MA，该基因的成功表达为进一步研究其抗病毒活性、抗病毒机理及结构-功能研究打下基础。
IM: The mutated gene of CA-MA Hybrid Peptide was constructed by PCR based point mutagenesis method, and expressed in E.coli. METHODS : A one step polymerase chain reaction (PCR) was used for the point directed mutation. The primers (P1, P2) were designed according to the sequence encoding CA-MA Hybrid Peptide, and mismatch was introduced into P1 with TGG for AGT substitution at position 16. PCR product of W16-CA-MA gene was performed in a one step PCR,and inserted into pGEX-4T-1 vector, and expressed in E.coli. RESULTS : DNA sequencing results show that the expected site mutations. Mutant was highly expressed in E.coli. CONCLUSION: A mutant peptide W16-CA-MA was been successful constructed. The successful expression of the gene will lay the foundation for further study of its antiviral activity, anti-virus mechanism and structure - functional studies.
韩跃武、冯惟萍、任慧子、陈建国
分子生物学生物工程学
天蚕素A-蛙皮素杂合肽聚合酶链反应定点突变融和表达
cecropin A (1–8)-magainin (1–12) (termed CA-MA) hybrid peptidepolymerase chain reaction ( PCR )rite-directed mutagenesiexpression
韩跃武,冯惟萍,任慧子,陈建国.天蚕素 A - 蛙皮素杂合肽基因突变体的构建及其表达[EB/OL].(2008-01-17)[2025-08-11].http://www.paper.edu.cn/releasepaper/content/200801-549.点此复制
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