Shiva-1a基因在杆状病毒中的表达及其表达产物抑菌活性的检测

Expression of the Shiva-1a Gene in Recombinant Baculovirus System and Analysis of the Peptide Antimicrobial Activity

根据GenBank上公布的shiva-1a的成熟肽基因序列,人工合成shiva-1a基因并加入6×His标签。将shiva-1a基因克隆到杆状病毒转座载体pBacFast-Dual中,筛选重组质粒,转化大肠杆菌DH10Bac感受态细胞,经蓝白斑筛选得到重组杆状病毒DNA,以脂质体介导法转染Sf-9昆虫细胞,待细胞出现病变后,收集上清液从而获得重组杆状病毒。用此杆状病毒感染的Sf-9细胞,Western blot检测出分子量约5 ku的shiva-1a多肽,与预期结果大小一致。体外抑菌试验证明,表达的shiva-1a多肽对大肠杆菌(DE3菌株)具有抑菌活性。

Based on the sequences of shiva-1a lytic peptide registered in GenBank,the fragment of the preferred codons of insect,shiva-1a gene sequence was designed and artificial synthesized,and then cloned into the pBacFast Dual vector of BAC-TO-BACTM recombinant baculovirus expression system.The recombinant plasmid was transformed into DH10Bac competent cell.By transfecting spodoptera fragiperda 9（Sf-9） cells with cellfectin and prepared recombinant bacmid,the recombinant baculovirus from the supernatant was obtained.PCR showed that the recombinant baculovirus was constructed successfully.Western blot showed that shiva-1a peptide with a molecular weight of 5 ku was expressed.In vitro antimicrobial test showed that shiva-1a antimicrobial had activity against E.coli.