CAJ | 학술논문

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 273 bp的完整ORF,序列分析表明与其它已报道的AgB8/2 cDNA序列的同源性在 99%～100%之间.优化的表达条件为,当菌液 OD_(600)值为0.6时,加入终浓度为0.05 mmol/L的 IPTG,37℃,振荡培养5 h.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.
지재극륭세립극구유 AgB8/2기인,우화원핵표체체계,감정순화단백병초보학정기진단개치.채용 RT-PCR확증 AgB8/2기인 cDNA,구건원핵표체재체pET-AgB8/2병재대장간균 BL21(DE3)중표체,우화표체조건.순화적AgB8/2융합단백경 Western blot감정정학후,채용반점면역금삼려법초보험증기혈청학진단개치.결과현시,극륭적 AgB8/2기인포함료 273 bp적완정ORF,서렬분석표명여기타이보도적AgB8/2 cDNA서렬적동원성재 99%～100%지간.우화적표체조건위,당균액 OD_(600)치위0.6시,가입종농도위0.05 mmol/L적 IPTG,37℃,진탕배양5 h.순화적단백경 Western blot감정위목적단백.극륭적 AgB8/2기인고도보수,원핵표체재체pET-AgB8/2구건정학병고효표체가용성융합단백,가작위특이성항원응용우반점면역금삼려법검측혈청항체.
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 AgB8/2 gene sequences released in GenBank. The highest expression level was achieved by inducing the bacteria with 0.05 mM IPTG, at 0.6 of OD_(600) 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 AgB8/2 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.