家蚕抗菌肽CD高效表达及其抗BmNPV感染作用

High-level Expression of Silkworm Antimicrobial Peptide and Its Anti-infection to BmNPV

通过大肠杆菌JM109诱导家蚕,提取其脂肪体总mRNA后,通过RT-PCR得到cDNA,根据GenBank上家蚕抗菌肽CecropinD的cDNA序列,设计并合成引物,然后PCR扩增得到CecropinD肽基因并克隆到pGEM-T载体中,经过EcoRΙ和XhoI酶切,连接并将CecropinD肽基因插入pET32a表达载体中。用重组质粒pET32a-ecropinD转化大肠杆菌BL21(DE3),在IPTG诱导下,融合蛋白Trx-CecropinD以可溶形式得到高效表达,经SDS-PAGE检测显示分子量为23kDa与预期大小相符,表达量约为总蛋白的30%。融合蛋白经Ni2+柱纯化后通过肠激酶切割后释放为Trx(18kDa)和CecropinD(5kDa),最后通过超滤管分离得到重组抗菌肽。通过抑菌实验测得重组CecropinD对于革兰氏阴性及阳性菌均有抑菌活性。并将重组CecropinD与家蚕病毒BmNPV作用混合4h后,一起投喂家蚕,发现病毒感染力有明显降低,说明其有抗病毒感染作用。

The peptide gene of Cecropin D was amplified by RT-PCR, cloned into the expression vector pET32a to generate the recombinant plasmid pET32a-CD. E.coli BL21 （DE3） were transformed by the plasmid and it was observed that the target gene was expressed at high-level in the form of fusion protein when induced with IPTG The molecular weight of the fusion protein was 23kDa by SDS-PAGE and it constituted about 30% of total cell proteins, indicative of high levels of expression. The fusion Trx-CD was purified by Ni-NTA chromatography, and then cleaved into two parts, Trx （18kI）a） and mature antimicrobial peptide CD（5kDa） by the enterokinase. The antim icrobial activity of the recombinant CD was checked by bacteria growth inhibition zone assay. Also, when BmNPV was mixed with CD for 4 h, the viral infectivity was markedly decreased. This indicated that the recombinant antimicrobial peptide CD has an inhibitory effect on BmNPV.