抑制新城疫病毒繁殖的九肽及其突变体基因的克隆与原核表达

目的克隆并表达抑制新城疫病毒(NDV)繁殖的九肽(Nonapeptide)及其突变体基因。方法设计并合成Non-apeptide2串联体及其突变体基因,克隆于质粒pUC18,经酶切回收目的基因,与经相同酶切的表达载体pGEX-4T-1连接,转化大肠杆菌BL21(DE3),筛选阳性菌落,抽提质粒进行鉴定。IPTG诱导表达后,进行SDS-PAGE及Western blot鉴定。结果重组表达质粒经双酶切、PCR及测序鉴定,证明构建正确。阳性重组菌的诱导表达产物经SDS-PAGE分析,在相对分子质量约29000处可见一明显条带,与理论值相符。Nonapeptide 2及其突变体蛋白的表达量分别占菌体总蛋白的41%和37%。Western blot结果显示两种蛋白均具有良好的反应原性。结论已成功克隆并表达了抗NDV繁殖的Nonapeptide及其突变体基因。

Cloning and Prokaryotic Expression of Genes Encoding Nonapeptide Inhibiting Newcastle Disease Virus Proliferation and Its Mutant

Objective To clone and express the genes encoding nonapeptide and its mutant, both inhibiting the proliferation of Newcastle disease virus (NDV). Methods Design and synthesize the two-copy concatemer of genes encoding nonapeptide and its mutant and clone into vector pUC18. Digest the constructed recombinant plasmids pUC-Nonapeptide2-1 and pUC-Nonapeptide2-2 with restriction endonuclease, and insert the obtained target genes into expression vector pGEX-4T-1. Transform the constructed recombinant plasmids pGEX-Nonapeptide2-1 and pGEX-Nonapeptide2-2 to E.coli BL21 (DE3) for expression under induction of IPTG. Identify the expressed products by SDS-PAGE and Western blot. Results Recombinant plasmids pGEX-Nonapeptide2-1 and pGEX-Nonapeptide2-2 were constructed correctly as proved by restriction analysis, PCR and sequencing. The expressed Nonapeptide2 protein and its mutant contained 41% and 37% of total somatic protein respectively. Each of the expressed protein showed a single band with relative molecular mass of about 29 000 on SDS-PAGE profile, which was consistent with the theoretical value, and showed good reactogenicity as proved by Western blot. Conclusion The genes encoding nonapeptide inhibiting NDV proliferation and its mutant were successfully cloned and expressed.