OC-IΔD86 (Oryzacystatin-IΔD86)基因的原核表达及活性分析

根据OC-IΔD86基因序列, 设计合成了7条寡核苷酸片段, 通过重叠延伸PCR技术合成了OC-IΔD86基因, 利用设计好的BamH I/ Xho I酶切位点将OC-IΔD86基因克隆到原核表达载体pet21b中, 在1 mmol/L的IPTG 诱导后5 h, OC-IDD86融合基因在大肠杆菌中得到表达, 表达产物处于可溶状态, 其表达量占总蛋白的11.4%, 可溶性蛋白的16.4%; 利用Ni-NTA系统纯化该蛋白并经PEG20000浓缩后, 活性分析表明该蛋白酶抑制剂在体外表现出对木瓜蛋白酶明显的抑制作用。这为转OC-IΔD86基因的抗根结线虫植物基因工程抗体制备, 以及进一步体内的抗根结线虫研究奠定了基础。

Prokaryotic expression of OC-IdeltaD86 (Oryzacystatin-IdeltaD86) gene and analysis of its activity

According to the amino acids sequence of OC-IdeltaD86 gene and Escherichia coli codon usage, we synthesized this gene by overlap extension PCR method with 7 oligonucleotides DNA fragments. The PCR fragment was inserted into pGEM-T-easy vector and the recombined plasmid was named pGEM-T-OC-IdeltaD86. Two oligonucleotides into which the BamH I and Xho I sites were introduced were designed and synthesized based on pGEM-T-OC-IdeltaD86 and pet21b, and the PCR fragment into which the BamH I and Xho I sites were introduced was obtained. After digesting it with BamH I and Xho I, OC-IdeltaD86 gene was cloned into the corresponding sites of pet21b and obtained prokaryotic expression vector pet21b-OC-IdeltaD86. OC-IdeltaD86 gene was expressed in E. coli (BL21(DE3)plysS) after IPTG(Isopropyl beta-D-1-thiogalactopyranoside) inducement for 5 hours. The fusion protein of OC-IdeltaD86:6His gene accounted for 11.4% of total protein and 16.4% of soluble protein, which had been successfully purified by Ni-NTA and concentrated by PEG20000. This protein can effectively inhibit papain activity in vitro and may be used in anti-nematode research in vivo.