NoV P颗粒嵌合PEDV S1 基因多表位抗原的制备

Preparation of NoV P-particle chimeric PEDV S1 gene multi-epitope antigen

为制备诺如病毒(Norovirus,NoV)P颗粒嵌合猪流行性腹泻病毒(Porcine Epidemic Diarrhea Virus,PEDV)S1基因多表位的抗原.本研究通过设计合成PEDV S1多表位基因序列,酶切后连接到诺如病毒P基因重组pGEX-4T-1载体,将载体转化至大肠杆菌DH5α感受态细胞后,提取质粒进行双酶切验证和测序.将测序正确的重组质粒转化至BL21表达菌体,使用不同浓度异丙基-β-D-硫代半乳糖苷(Isopropyl-β-D-thiogalactopyranoside,IPTG)在不同诱导时间下进行原核表达,确定最佳诱导条件,并对重组蛋白进行可溶性分析.使用鼠源谷胱甘肽-S-转移酶(Glutathione-S-transferase,GST)单抗和PEDV阳性血清通过蛋白免疫印迹试验检测纯化后的重组蛋白.重组质粒NoV P-partical-PEDV-SE经双酶切鉴定获得大小约510 bp的目的基因条带,进一步测序结果证明重组质粒构建成功.诱导表达的重组蛋白分子质量约为79 ku,与预期大小一致.使用1.0 mmol/L IPTG在37℃条件下培养2 h作为最佳诱导条件,对超声破碎后的菌体进行检测,试验结果显示重组蛋白可在上清液中表达.鼠源GST单抗与PEDV阳性血清均能特异性地识别纯化后的重组蛋白,表明该重组蛋白具有良好的反应原性.本研究通过对NoV P-partical-PEDV-SE蛋白原核表达及纯化,成功获得大量纯度较高的重组蛋白,为进一步制备猪流行性腹泻多表位口服疫苗,实现对PEDV的有效预防奠定基础.

In order to prepare a multi-epitope antigen of Porcine epidemic diarrhea virus(PEDV)S1 gene chimerized with Norovirus(NoV)P particles.The PEDV S1 multi-epitope gene sequence was designed and synthesized,enzymatically ligated into the recombinant pGEX-4T-1 vector of the NoV P gene,transformed the vector into E.coli DH5αreceptor cells,and extracted the plasmid for double digestion verification and sequencing.The correctly sequenced recombinant plasmids were transformed into BL21-expressing organisms,and the recombinant proteins were analyzed for solubility using different concentrations of Isopropyl-β-D-thiogalactopyranoside(IPTG)at different induction times to determine the optimal induction conditions.Western blot detection of purified recombinant proteins using murine Glutathione-S-transferase(GST)monoclonal antibody and PEDV-positive serum.The recombinant plasmid NoV P-partical-PEDV-SE was identified by double digestion and a target gene band of approximately 510 bp in size was obtained.The molecular mass of the induced recombinant protein was about 79 ku,which was consistent with the expected size.The recombinant protein was expressed in the supernatant after sonication of the fragmented bacterium using 1.0 mmol/L IPTG at 37℃for 2 h as the optimal induction condition.Both murine-derived GST monoclonal antibody and PEDV-positive serum specifically recognized the purified recombinant protein,indicating that the recombinant protein had good reactogenicity.In this study,a large amount of recombinant protein with high purity was successfully obtained by in situ expression and purification of NoV P-partical-PEDV-SE protein,which is the basis for further preparation of a multi-epitope oral vaccine against PEDV in pigs.