中文摘要：

【目的】研究利用大肠杆菌可溶性表达PCV2b亚型ORF2基因,利用重组Cap蛋白制备PCV2 VLP疫苗的可行性。【方法】根据大肠杆菌密码子使用频率表优化合成PCV2 NJ株ORF2基因,将其插入原核表达载体pQZ1,鉴定阳性后转入表达宿主菌BL21,利用原核表达平台CVC1102对重组菌进行诱导表达。利用SDS-PAGE与Western blotting对目的基因的表达及产物的可溶性进行分析;利用电镜分析技术鉴定重组Cap蛋白VLP组装;利用BCA试剂盒测定重组大肠杆菌破碎后上清中总蛋白量,利用薄层扫描分析确定目的蛋白百分比,得出目的蛋白量,将重组Cap蛋白的浓度调整为1 mg.mL^-1。使用不同剂量的重组蛋白与206佐剂乳化,免疫2—3周龄抗原抗体双阴性的仔猪,同时设含免疫增强剂CVC1301、CVC1302的试验组、同类制品免疫对照组与空白对照组,免疫后14 d与21 d所有试验猪采血,分离血清,利用武汉科前生物科技有限公司的猪圆环病毒2型抗体ELISA检测试剂盒检测试验猪血清中PCV2抗体水平;免疫后28 d试验猪按照兽用生物制品质量标准汇编中公布的PCV2攻毒程序,利用PCV2 NJ株F6代进行强毒攻击试验,肌肉注射与口服PCV2 NJ株各10^5.0TCID50,同时利用乳化的钥匙孔血蓝蛋白及硫酸巯基乙醇培养基进行免疫刺激,攻毒后25 d,对所有试验猪进行解剖检测,通过攻毒过程中试验猪体温变化、试验猪平均相对日增重、解剖时试验猪肺部与相关淋巴结的大体变化及攻毒后PCV2核酸检测等4个方面评价大肠杆菌表达制备的PCV2 VLP的免疫效力。【结果】SDS-PAGE结果表明PCV2 NJ株优化的ORF2基因在大肠杆菌中得到了高效表达,表达产物以完全可溶性形式存在于菌体超声波破碎后的上清中;Western blotting分析结果显示表达的重组Cap蛋白能够与PCV2阳性血清发生反应;电镜下观察可见大量直径在17 nm左右的PCV2病毒样颗粒存在于超声破碎后的上清中;500μg/头?

英文摘要：

【Objective】The objective of this study is to develop recombinant virus like particles vaccine for Porcine circovirus type 2 through soluble prokaryotic expression of ORF2 gene. 【Method】 The optimized ORF2 gene of PCV2 NJ strain was synthesized according to codon usage of E.coli, and then the optimized ORF2 gene was cloned into p QZ1 to get the recombinant prokaryoticexpression vector named p QZ-Cap. The target gene was expressed with 1.0 mmol·L^-1 IPTG induction for 24 h under 15℃ on the prokaryotic expression platform in authors＇ laboratory following the recombinant plasmid p QZ-Cap transformed into host E.coli BL21. Firstly, SDS-PAGE and Western blotting were used to identify the expression and solubility of the recombinant protein.Secondly, assemble for recombinant Cap VLP was evaluated through electron microscope analysis technology. Furthermore, swines were inoculated with recombinant Cap protein emulisified in SPPEIC 206 adjuvant, and the immunogenicity of recombinant Cap protein vaccine was evaluated by PCV2 specific antibody detection and virus attack. 【 Result 】 SDS-PAGE and Western blotting results indicated that the optimized ORF2 gene of PCV2 NJ strain could be efficiently expressed in E.coli BL21 in form of completely soluble. Abundant PCV2 VLP with diameter about 17 nm could be observed through electron microscope, exist in supernatant of the induced E.coli after ultrasonication. VLP vaccine composed of recombinant Cap protein possessed satisfactory immunity. Swine immunized with this VLP vaccine generated perfect antibody response with 100% qualified at 21 d post one sole vaccination and could be completely protected from pathogenic virus attack.【Conclusion】To summarize, optimized ORF2 gene of PCV2 NJ strain was successfully expressed in E.coli completely soluble and recombinant Cap protein can auto-assemble into VLP particles after ultrasonication. The VLP particles pose perfect immunity and could be used as subunit vaccines to prevent PCV2 infection.