灰飞虱内生病毒HiPV外壳蛋白VP1多克隆抗体的制备及在病毒检测中的应用

【目的】前期发现水稻条纹病毒(rice stripe virus, RSV)可与介体灰飞虱Laodelphax striatellus体内的HiPV病毒(Himetobi P virus, HiPV)互作。本研究旨在制备HiPV外壳蛋白VP1的多克隆抗体，并评估其在HiPV病毒检测中的可用性，以为深入研究HiPV-RSV和HiPV-灰飞虱的互作机制提供技术支持。【方法】以RT-PCR方法从灰飞虱成虫体内扩增HiPV主要外壳蛋白基因VP1，然后将VP1基因亚克隆至原核表达载体pET-32a中，构建表达载体pET-VP1。将重组质粒转化大肠杆菌Escherichia coli BL21 (DE3)，经IPTG诱导、Ni²⁺-NTA亲和层析纯化，获得重组蛋白，免疫新西兰大白兔，制备抗体。【结果】从灰飞虱体内克隆到774 bp的HiPV外壳蛋白基因VP1，经原核表达、纯化，获得分子量约47.5 kD的融合蛋白，免疫新西兰大白兔后获得VP1多克隆抗体。该抗体间接ELISA效价达1∶819 200，与HiPV外壳蛋白VP1有特异性反应，而与灰飞虱蛋白无交叉反应。利用该多克隆抗体建立了检测单头灰飞虱成虫体内HiPV的Western blot和免疫捕获RT-PCR方法，检测结果显示HiPV在携带和不携带RSV的灰飞虱高亲和性群体内均广泛存在。【结论】利用制备的HiPV的VP1多克隆抗体可特异性检测灰飞虱体内HiPV。本研究为HiPV病毒的快速检测以及HiPV-RSV互作、HiPV-灰飞虱互作研究提供了技术支持。

Preparation of the polyclonal antibody of capsid protein VP1 of Himetobi P virus in the small brown planthopper,Laodelphax striatellus (Hemiptera: Delphacidae) and its application in the virus detection

【Aim】 In our previous work, the interaction between rice stripe virus (RSV) and Himetobi P virus (HiPV) of the small brown planthopper (SBPH), Laodelphax striatellus, was found. This study aims to prepare the polyclonal antibody (PAb) of the capsid protein VP1 of HiPV and to assess its application in the detection of HiPV, so as to provide a technical basis for further studying the interaction of HiPV-RSV and HiPV-SBPH. 【Methods】 VP1 gene of HiPV was amplified from SBPH adult via RT-PCR, and subcloned into the prokaryotic expression vector pET-32a to construct the vector pET-VP1. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3), and the recombinant protein was obtained by IPTG induction and purified by Ni²⁺-NTA affinity chromatography. The antibody was prepared by immunizing New Zealand white rabbit with the purified recombinant protein. 【Results】 A 774 bp product of capsid protein gene VP1 of HiPV was amplified from SBPH, and a 47.5 kD VP1 fusion protein was obtained through prokaryotic expression and purification. The purified VP1 recombinant protein was used to immunize New Zealand white rabbit, and the PAb was prepared. The titer of PAb was 1∶819 200 in indirect ELISA, and the PAb could react specifically with HiPV VP1 but not with SBPH proteins. Using PAb against HiPV, methods of Western blot and immunocapture-RT-PCR (IC-RT-PCR) for the detection of HiPV in single adult of SBPH were established. The detection results showed that HiPV was prevalent in the RSV-infected and uninfected SBPH high-affinity populations. 【Conclusion】 HiPV can be detected specifically in SBPH using the prepared VP1 polyclonal antibody. This study provides a technical support for the rapid detection of HiPV and the study of HiPV-RSV and HiPV-SBPH interaction.