香蕉线条病毒 MP 功能域基因的克隆、原核表达及抗血清制备
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国家自然科学基金(30671358);广东省自然科学基金(C036845)


Prokaryotic expression and antiserum preparation for functional domain of Banana streak virus movement protein gene
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    【目的】制备香蕉线条病毒广东分离物(BSV-GD)MP功能域基因编码蛋白的多克隆抗血清,为BSV基因编码蛋白功能的研究提供条件.【方法】对BSV-GD ORF3基因氨基酸序列进行生物信息学分析,得出MP功能域基因序列,克隆该基因并插入载体pET-28b(+)构建原核表达载体,经IPTG诱导表达后,采用超声波裂解法进行蛋白可溶性分析,利用组氨酸标签纯化试剂盒对目的融合蛋白进行纯化和回收,然后以纯化的目的蛋白为抗原免疫健康家兔制备其多克隆抗血清;通过Western-blot分析该抗血清的特异性,间接ELISA法检测该抗血清的效价.【结果和结论】MP功能域基因在ORF3中的序列为61~311 aa处,核酸序列长753 bp. 试验克隆了该基因并成功构建了其原核表达载体pET28b-MP,经IPTG诱导1 h后表达了相对分子质量约为30 800的融合蛋白6His·MP.可溶性分析表明该融合蛋白以包涵体形式存在,纯化获得了高纯度的目的融合蛋白,以其为抗原成功制备了BSV-GD MP功能域基因编码蛋白的多克隆抗血清;分析表明该抗血清具有很强的特异性,其效价高达204 800倍以上.

    Abstract:

    【Objective】To provide antibody of Banana streak virus Guangdong isolate (BSV-GD) and to provide references for the function research of proteins encoded by BSV ORF3 further. 【Method】The MP functional gene sequence was obtained through bioinformatics analysis. The gene was cloned and inserted into the plasmid pET-28b(+) to construct the prokaryotic expression recombinant plasmid. The recombinant vector was induced by IPTG to express the fusion protein 6His·MP. Soluble analysis of the fusion protein was carried out by ultrasonic lysis method. The highly purified protein was obtained by His-tag purification kit. The special polyclonal antibody was generated by immunizing healthy rabbit using the purified protein as antigen. The specificity of antiserum was detected by Western-blot. The antibody titer was determined by indirect enzyme immunoassay. 【Result and conclusion】The analysis showed that the MP functional gene was 61-311 aa of the ORF3 sequence and the nucleotide sequence length was 753 bp. The prokaryotic expression recombinant plasmid pET28b-MP was constructed, and the expressed fusion protein 6His·MP was about 30 800 in size. Soluble analysis of the fusion protein indicated that it was an inclusion body. The highly purified target protein was obtained. The special polyclonal antiserum was generated according to the purified protein. The assay suggested that the antiserum had very strong specificity, and the antibody titer was higher than 1∶204 800.

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陈 秀,饶雪琴,阮小蕾,李华平.香蕉线条病毒 MP 功能域基因的克隆、原核表达及抗血清制备[J].华南农业大学学报,2014,35(2):47-52

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  • 收稿日期:2013-04-16
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  • 在线发布日期: 2014-02-28