猴D型逆转录病毒p27基因的克隆表达及诊断价值评价

doi:10.3969/j.issn.1674-5817.2016.04.005

实验动物与比较医学 ›› 2016, Vol. 36 ›› Issue (4): 270-275.DOI: 10.3969/j.issn.1674-5817.2016.04.005

猴D型逆转录病毒p27基因的克隆表达及诊断价值评价

周洁¹, 杨燕飞^1,2, 胡建华¹, 陶凌云¹, 高诚¹

1.上海实验动物研究中心, 上海201203;
2.扬州大学兽医学院, 扬州225009

出版日期:2016-08-25 发布日期:2016-08-25
作者简介:周洁(1978-), 女, 博士, 副研究员, 主要从事动物病毒分子生物学及免疫学方面的研究。E-mail: zhoujie0526@163.com
基金资助:
上海市科委科技创新行动计划(13140900600), 上海市科委研发平台专项(15DZ2292400)

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

ZHOU Jie¹, YANG Yan-fei^1,2, HU Jian-hua¹, TAO Ling-yun¹, GAO Cheng¹

1. Shanghai Research Center of Laboratory Animal, Shanghai 201203;
2. Yangzhou University College of Veterinary, Yangzhou 225009

Online:2016-08-25 Published:2016-08-25

摘要/Abstract

摘要： 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4 h,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。

关键词: 猴D型逆转录病毒, p27基因, 原核表达, 血清学检测

Abstract: Objective To Clone and express p27 gene of simian type-D retrovirus and evaluate its diagnostic potential. Method The p27 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.

Key words: Simian type-D retrovirus, p27 gene, Prokaryotic expression, Serological detection

中图分类号:

Q95-33

周洁, 杨燕飞, 胡建华, 陶凌云, 高诚. 猴D型逆转录病毒p27基因的克隆表达及诊断价值评价[J]. 实验动物与比较医学, 2016, 36(4): 270-275.

ZHOU Jie, YANG Yan-fei, HU Jian-hua, TAO Ling-yun, GAO Cheng. Production of Recombinant Simian Type-D Retrovirus p27 Gene and Evaluation of its Diagnostic Potential[J]. Laboratory Animal and Comparative Medicine, 2016, 36(4): 270-275.

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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

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