天蚕抗菌肽AD基因在AcNPV载体系统中的表达研究

Study on expression of cecropin AD gene in AcNPV vector expression system

目的 研究利用昆虫杆状病毒载体系统在昆虫培养细胞和虫体中高效表达新型多肽抗生素天蚕抗菌肽AD(cecropinAD ,CAD)。方法 采用聚合酶链反应 ( polymerasechainreaction ,PCR)点突变技术改造CAD基因 ,改造后的CAD基因先克隆到质粒 pGEM Teasyvector中进行鉴定和序列分析 ,然后再亚克隆到苜蓿丫纹夜蛾核型多角体病毒 (Autographacalifornicanuclearpolyhedrosisvirus ,AcNPV)表达载体pAcGP67B中 ,获得重组病毒载体 pAcAD。用重组病毒载体 pAcAD和野生AcNPVDNA共感染草地夜蛾 (Spodopterafrugiperdoda)Sf9细胞 ,经空斑法筛选得到重组病毒AcNPVAD。重组病毒AcNPVAD经PCR扩增鉴定 ,证实了CAD基因已经整合到重组病毒AcNPVAD中。结果 以AcNPVAD转染Sf9细胞和苜蓿丫纹夜蛾幼虫 ,抑菌活性试验证实CAD基因在昆虫培养细胞Sf9和苜蓿丫纹夜蛾幼虫虫体中均得到表达 ,表达产物具有抑菌作用。通过离子交换层析 ,杂蛋白基本去除 ,重组抗菌肽获得初步纯化 ,重组抗菌肽活性峰位于 0 75mol/L处。结论 CAD基因在昆虫杆状病毒载体系统获得了表达 ,表达产物具有抑菌活性。

Objective To study cecropin AD (CAD) gene expression in culture Sf9 cell and insect by baculovirus vector expression system. Methods CAD gene was modified by PCR amplification. The Modified CAD gene was first cloned into plasmid pGEM T easy vector for DNA sequencing, then was subcloned into Autographa californica nuclear polyhedrosis virus (AcNPV) vector pAcGP67B to obtain recombinant virus vector PacAD. Spodoptera frugiperododa Sf9 cells was coninfected with pAcAD and wild type AcNPVDNA, and recombinant virus AcNPCAD was obtained through sea plaque selection. By PCR amplification analysis, CAD gene was proved to be integrated into recombinant virus AcNPVAD. Results Recombinant virus AcNPVAD was transinfected Sf9 cell and Autographa californica young larvae, CAD gene have been confirmed to be expressed in Sf9 cell and Autographa californica young larvae by determination of antibacterial activity. Through CM Sephrose FF column chromatogram, recombinant antibacterial peptide was purified, antibacterial active peak was located at 0 75mol/L eluting concentration. Conclusion Cecropin AD gene was expressed in baculovirus vector expression system and expressed product had antibacterial activity.