抗菌肽MSI-78基因在大肠埃希菌DH5α中的克隆及阳性重组体鉴定

Cloning of MSI-78 Gene in Escherichia coli DH5α and Identification of Positive Recombinant

目的研究抗菌肽MSI-78基因的克隆及鉴定方法,为抗菌肽MSI-78基因的表达及抗菌活性研究奠定基础。方法根据MSI-78氨基酸序列及大肠埃希菌(ECO)偏爱密码子,推出MSI-78编码基因,化学合成获得MSI-78基因,ECORⅠ和PstⅠ消化后与pUC18载体连接并转化大肠埃希菌DH5α,α-互补筛选阳性重组菌,单酶切、双酶切、PCR鉴定阳性重组体。结果通过ECORⅠ单酶切、ECORⅠ和PstⅠ双酶切抽提的阳性重组质粒,证实阳性重组质粒中含有抗菌肽MSI-78基因,特异性引物PCR扩增也证实阳性重组质粒中含有抗菌肽MSI-78基因,说明MSI-78基因成功在大肠埃希菌DH5α克隆。结论MSI-78基因的克隆为后期的表达、纯化及生物活性研究奠定了基础。

OBJECTIVE To clone the MSI-78 gene for the purpose of providing evidence for further studies in prokaryotic expression and activities of antimierobial peptides. METHODS According to the amino acid sequences of MSI-78, the MSI-78 gene was designed favorable for the Escherichia coli codons. After EcoR Ⅰ and Pst Ⅰ disgestion, cohesive ends were added to both ends respectively and the MSt-78 gene was synthesized by chemical methods. Then, the MSI-78 gene was ligated with pUC-18,transformed into the E. coli DH5α. Through filtration of α complementary screening, the positive recombinant was finally identified by enzyme digestion of ECOR Ⅰ and ECOR Ⅰ/Pst Ⅰ and by PCR. RESULTS The MSI-78 gene was ligated with pUC-18 and transformed into the E. coli DH5a. As a result, MSI-78 gene was cloned in 17,. coli DH5a successfully. CONCLUSIONS The cloning of the MSI-78 gene provides evidence for further studies of its prokaryotic expression and activities of antimierobial peptides.