家蚕杆状病毒多重展示系统的构建及其应用于多价疫苗的模型研究

Baculovirus display system (BDS) can be used to develop vaccine through displaying target antigens on the envelope surface of baculovirus. However, traditional BDS has many disadvantages, such as low efficiency of display, few in number of displayed antigens, and high cost of insect cells culture, which greatly impede the application of BDS. This project plans to construct a new efficient, inexpensive silkworm-baculovirus multigene display system (SBMDS) on the base of silkworm-baculovirus multigene expression system (SBMES) formed by our group. The designed system is able to efficiently display 10 target antigens on the outer surface of virus envelope, supplying a nice technique platform for the production of animal polyvaccines. SBMDS can not only display multi antigens, but also display immunologic enhance factors, immunologic escape factors, and even immunologic adjuvants to provide higher immune efficiency. Here, we choose swine influenza virus (SIV) as an experimental model, to construct a multigene chimeric display virus containing the hemagglutinin (HA) and neuraminidase (NA) of SIV-H1N1, the NA of SIV-H1N2, the HA of SIV-H3N2, the invasion and the decay-accelerating factor (DAF) of mammalian cell, and detect its immunogenicity. This research has great advantages on the high efficiency of multigene display and the low cost of silkworm feed. It will provide strong base for the building of polyvaccine platform, as well as the preventing of animal zymosis.

杆状病毒表面展示系统(BDS)可通过将目标抗原展示在病毒粒子囊膜表面用于疫苗开发，但传统BDS存在展示效率不高，展示目标因子过少，细胞培养昂贵等缺点而无法满足现代疫苗发展需要。本项目拟以课题组开发的家蚕杆状病毒多基因表达系统为基础，构建出一套高效经济的新型家蚕杆状病毒多基因展示系统(SBMDS)。该系统能将多达10个目标基因展示于病毒囊膜外表面，可展示多个抗原，也可同时展示免疫增强因子、免疫逃避因子、甚至蛋白免疫佐剂，以显著提高免疫效率，从而为发展高效多价动物疫苗提供平台。如以猪流感病毒(SIV)为模型，通过构建同时展示SIV的血球凝集素(HA)、唾液酸苷酶(NA)及哺乳动物细胞侵袭素和免疫逃避因子等多种蛋白的展示病毒，并确定其免疫原性，为今后发展出有效防治SIV疫苗奠定基础。本研究充分发挥多基因展示的效能优势和家蚕饲养的成本优势，在新型疫苗研发平台构建和预防动物传染等方面具有重要意义。

传统BDS由于缺乏合适的穿梭载体、存在蛋白竞争和表达时序性差异，容易导致外源蛋白展示效率低、种类少。利用over-lap PCR等技术，成功构建了2种可应用于细菌内同源重组的基因片段TB1-Zeocin2R-ph-TB2和gp64F-Zeocin1R-gp64R，利用电转化和细菌内同源重组等技术，首次成功构建出2种经济高效的新型SBMDS，该重组杆状病毒表达量高、毒力强。双荧光素酶融合展示等研究结果显示基于TB1-Zeocin2R-ph-TB2的SBMDS可将外源基因展示效率提高约0.8倍，且操作便捷；同时，ires介导的GP64低量表达时，基于gp64F-Zeocin1R-gp64R的SBMDS可将外源基因展示效率提高约4倍。. 成功构建egfp单基因展示病毒AcBV-G-GP64-I64、egfp+mCherry双基因共展示病毒AcBV-G-M-GP64-I64和egfp+mCherry+eyfp三基因共展示病毒AcBV-G-M-Y-GP64-I64。CLSM和Western blot鉴定结果3种荧光蛋白成功组装到重组杆状病毒，并可稳定、高效地展示在细胞膜表面。. 应用零背景Tn7转座和cre-LoxP特异性位点重组技术，将SIV、VSV、PCV2、PRRSV或CSFV等畜牧业常见病毒囊膜蛋白和Invasin侵染蛋白C端截短片段等重组至SBMDS内，成功构建出4基因（egfp+mCherry+eyfp+vsv-g）和6基因（egfp+mCherry+gp5+pcv2+e2+inV）共展示重组杆状病毒AcBV-Zeo-Δgp64-HG-FM-SY-V-I64、AcBV-p64spFP64-p64spSI64-p64spCE64-p64spHG64-phG-phM。重组病毒的毒力测定和融合蛋白鉴定表明，外源蛋白成功组装至子代病毒。PCV2-Cap、CSFV-E2和PRRSV-GP5的ELISA测定结果表明：6基因共展示重组病毒对PCV2、CSFV和PRRSV等免疫效果良好。gp5、pcv2、e2、inV和vsv-g等Coden usage比对分析表明均可在SBMDS中获得较好表达效果。. 新型SBMDS平台可为多基因共展示、抗原呈递和蛋白互作等抗病育种和分子机制相关研究奠定理论基础。