水貂肠炎病毒VP2基因原核表达及病毒样颗粒制备

doi:10.16431/j.cnki.1671-7236.2021.02.004

摘要/Abstract

摘要： 试验旨在制备水貂肠炎病毒（Mink enteritis parvovirus，MEV）可溶性VP2蛋白及其病毒样颗粒（virus-like particles，VLPs）。优化并合成MEV VP2基因，克隆至原核表达载体pET-30a（+），将重组质粒pET-30a-VP2与分子伴侣pTf16共同转化到宿主菌ER2566中，以不同培养温度、L-阿拉伯糖浓度和IPTG浓度进行诱导表达，收集样品进行SDS-PAGE和Western blotting分析。通过硫酸铵沉淀结合蔗糖密度梯度离心法对表达产物进行纯化，由SDS-PAGE进行鉴定，透析去除蔗糖，通过动态光散射技术（DLS）和透射电子显微镜（TEM）观察不同组装条件下VLPs的粒径和形态。用制备的MEV VLPs疫苗免疫水貂评价其免疫原性。结果显示，以2 g/L L-阿拉伯糖、0.2 mmol/L IPTG、25 ℃培养16 h为诱导条件时，VP2蛋白在大肠杆菌中可溶性表达，分子质量约为65 ku。经Western blotting鉴定VP2蛋白具有良好的抗原特异性。将此表达产物利用硫酸铵沉淀结合蔗糖密度梯度离心法纯化后，纯度可达90%以上。在pH 8.0，150 mmol/L NaCl的透析液中，VP2经自组装可形成与天然MEV形状和粒径相似且具有血凝特性（1：2¹⁴）的VLPs。VLPs疫苗免疫水貂21 d后，测得水貂血清中的血凝抑制（HI）抗体水平均相对最高，可达1：2¹¹，说明该VLP疫苗有较好的免疫原性，能有效预防MEV的感染。本试验结果表明，将pET-30a-VP2与pTf16在原核表达系统中共表达，可获得具有高免疫原性的VLPs，为后期MEV VLPs疫苗的研发奠定基础。

关键词: VP2; 原核表达; pTf16; 病毒样颗粒

Abstract: This study was aimed to produce soluble protein of Mink enteritis parvovirus (MEV) capsid protein VP2 and to obtain virus-like particles (VLPs) of MEV.The MEV VP2 gene was optimized,synthesized and cloned into prokaryotic expression vector pET-30a(+).The recombinant vector pET-30a-VP2 was expressed by co-transformation with pTf16 into ER2566,and was induced at different temperatures,L-arabinose concentrations and IPTG concentrations,and analyzed by SDS-PAGE and Western blotting.VP2 was purified by ammonium sulfate precipitation combined with sucrose density gradient centrifugation,then identified by SDS-PAGE.Under the different assembled conditions,the purified protein was self-assemble into VLPs with the removal of sucrose.VLPs were analyzed by dynamic light scattering (DLS) and transmission electron microscopy (TEM).Minks were immunized with homemade MEV VLPs vaccine to identify their immunogenicity.The results showed that recombinant VP2 protein was highly expressed in the supernatant of E.coli after induction with 2 g/L L-arabinose and 0.2 mmol/L IPTG at 25 ℃ for 16 h.SDS-PAGE showed that the fusion protein was about 65 ku.Western blotting confirmed that VP2 had good antigen specificity.VP2 was purified by ammonium sulfate precipitation combined with sucrose density gradient centrifugation and degree of purity could reach more than 90%.DLS and TEM results indicated that VLPs had similar size,shape and hemagglutination (1:2¹⁴) with the authentic virus capsid at 150 mmol/L NaCl,pH 8.0.Immunization with VLPs vaccine could induce high-titer hemagglutination inhibition antibody (the highest was 1:2¹¹) in minks after 21 days,indicating that the VLP vaccine had good immunogenicity and could effectively prevent MEV.VLPs with high immunogenicity could be obtained by expression of VP2 with pTf16 in prokaryotic expression system,which laid a foundation for further research on MEV VLPs vaccine.

Key words: VP2; prokaryotic expression; pTf16; virus-like particles

中图分类号:

S855.3

吴铭洁, 夏霖亚, 王蕾, 张宁, 罗国良, 殷玉和, 吴丛梅. 水貂肠炎病毒VP2基因原核表达及病毒样颗粒制备[J]. 中国畜牧兽医, 2021, 48(2): 433-442.

WU Mingjie, XIA Linya, WANG Lei, ZHANG Ning, LUO Guoliang, YIN Yuhe, WU Congmei. Prokaryotic Expression of Mink Enteritis Parvovirus VP2 Gene and Assembly of Virus-like Particles[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(2): 433-442.

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