细粒棘球蚴AgB8/2基因的原核表达及免疫学鉴定

Prokaryotic Expression and Immunologic Identification of AgB8/2 Gene from Echinococcus granulosus

旨在克隆细粒棘球蚴AgB8/2基因,优化原核表达体系,鉴定纯化蛋白并初步确定其诊断价值。采用RT-PCR扩增AgB8/2基因cDNA,构建原核表达载体pET-AgB8/2并在大肠杆菌BL21（DE3）中表达,优化表达条件。纯化的AgB8/2融合蛋白经Western blot鉴定正确后,采用斑点免疫金渗滤法初步验证其血清学诊断价值。结果显示,克隆的AgB8/2基因包含了273bp的完整ORF,序列分析表明与其它已报道的AgB8/2cDNA序列的同源性在99%-100%之间。优化的表达条件为,当菌液OD600值为0.6时,加入终浓度为0.05mmol/L的IPTG,37℃,振荡培养5h。纯化的蛋白经Western blot鉴定为目的蛋白。克隆的AgB8/2基因高度保守,原核表达载体pET-AgB8/2构建正确并高效表达可溶性融合蛋白,可作为特异性抗原应用于斑点免疫金渗滤法检测血清抗体。

It aimed at cloning the AgB8/2 gene from Echinococcus granulosus and expressing fusion protein in E. coli BL21 （ DE3 ） followed by immunologic identification with Western blot and diagnostic value with dot immunogold filtration assay （ DIGFA ）. AgB8/2 gene was amplified from Echinococcus granulosus by RT-PCR, and then was cloned into pET-44a（ ＋ ）to construct the prokaryotic expression vector pET-AgB8/2. AgB8/2 fusion protein was expressed successfully in E. coli BL21 （DE3）at an optimized expression condition and then was purified after identification by SDS-PAGE and Western blot. DIGFA was used to value serological significance of the protein. Results showed that the cloned AgB8/2 gene sequence includes 273 bp ORF and has 99% - 100% identity with other AgBS/2 gene sequences released in GenBank. The highest expression level was achieved by inducing the bacteria with 0.05 mM IPTG, at 0.6 of OD600 value for 5 h at 37℃. Purified soluble fusion protein was identified to be the correct target protein by SDS-PAGE and western blot. Thus,it proved that The AgBS/2 gene is highly conservative. The prokaryotic expression vector pET-AgB8/2 was constructed successfully and expressed at a high level under the optimized expression condition. The fusion protein can be used to dot immunogold filtration assay as a specific antigen.