猴D型逆转录病毒p27基因的克隆表达及诊断价值评价

Production of Recombinant Simian Type-D Retrovirus p27 Gene and Evaluation of its Diagnostic Potential

目的 克隆表达猴D型逆转录病毒（SRV） p27基因,评价其诊断价值.方法 PCR扩增p27基因片段,定向克隆至原核表达载体pET-28b（＋）,重组质粒转化大肠杆菌Rosetta （DE3）中诱导表达,利用SDS-PAGE和Western blot对表达产物进行鉴定,目的蛋白经镍柱亲和层析纯化后用ELISA方法评价其诊断价值.结果 重组质粒转化宿主菌后在37℃,异丙基-β-D-硫代半乳糖苷（IPTG）浓-度为0.5 mmol/L的条件下诱导4h,可溶性p27蛋白表达量最大,且具有免疫学活性.纯化后测定融合蛋白浓度为2.043 g/L,纯度93.3％,以融合蛋白为诊断抗原包被ELISA板检测3份标准阳性血清和22份阴性血清,检出率为100％.结论 p27基因成功表达并具有良好的反应原性,可作为猴D型逆转录病毒血清学检测的候选抗原.

Objective To Clone and express p27 gene of simian type-D retrovirus and evaluate its diagnostic potential. Method Thep27 gene was amplified by RT-PCR and cloned into the prokaryotic expressive vector pET-28b（＋） after sequencing. Recombinant plasmid was transformed into E.coli Rosetta DE3） and recombinant protein was analysed by SDS-PAGE and Western blot. The protein was purified affinity chromatography with Ni-NTA, and its diagnostic value was evaluated by ELISA. Results The highest soluble expression level of recombinant protein was obtained after being induced at 37 ℃ for 4 h, with the concentration of Isopropyl-β-D-thiogalactoside （IPTG） was 0.5 mmol/L, and the recombinant protein had immunological activity. After purified, the concentration of protein was 2.043 g/L, and the purity was 93.3%. The indirect ELISA was developed using the purified recombinant protein, detection of 3 standard positive sera and 22 negative sera showed the detection rate were 100%. Conclusion Recombinant p27 protein from prokaryotic expression system had perfect antigenicity, which would can be used as the candidate antigen of simian type-D retrovirus.