家畜口蹄疫新型疫苗研制与生产工艺创新2014年度报告

王永录 景志忠 李志勇 齐鹏 李祥敏

摘 要：正在研制的口蹄疫新型疫苗有10多种，分别是纳米微球黏膜免疫疫苗，表位肽疫苗、口蹄疫O型复合表位蛋白疫苗、A型重组毒株（Re-A/WH/2009）的口蹄疫O型、A型、亚洲Ⅰ型三价灭活疫苗、猪口蹄疫O型广谱基因工程病毒灭活疫苗、口蹄疫O型标记疫苗、猪O型Mya98毒株空衣壳疫苗、猪口蹄疫O型合成肽疫苗（多肽2600+2700+2800）、牛口蹄疫（O型、Asia1型）二价合成肽疫苗、以及口蹄疫病毒活载体疫苗。其中取得突破性进展的有5种，牛口蹄疫（O型、Asia1型）二价合成肽疫苗PD50值大于6.0，免疫持续期为6个月，保存期为12个月，已完成所有实验室研究和临床试验，申请了新兽药注册并已通过初审；猪口蹄疫O型合成肽疫苗（多肽2600+2700+2800）PD50值大于6.0，免疫持续期为6个月，保存期为12个月，已完成所有实验室研究和临床试验，现已申请新兽药注册并通过复核试验和复审；含A型重组毒株（Re-A/WH/2009）的口蹄疫O型、A型、亚洲Ⅰ型三价灭活疫苗PD50值大于6.0，免疫持续期为6个月，保存期为12个月，获得了农业部新兽药注册证书，实现了产业化；猪口蹄疫O型广谱基因工程病毒灭活疫苗已申报农业部临床试验批文；口蹄疫O型标记疫苗已完成疫苗质量研究，并已申请新兽药注册。其他疫苗研究也均取得了程度不等的进展。利用反向遗传操作技术平台，获得基因工程修饰病毒疫苗株5株：（1）rV-STN-5；（2）9O/rV-1；（3）Re-A/WH/2009；（4）Re-Mya/98/BY/2010；（5）Re-Asia1/HN/2006。利用基因克隆、重组等技术，获得其他基因工程修饰病毒9株，分别为：（1）表达O型FMDV不同亚型主要免疫原性基因的重组伪狂犬病毒1株；（2）共表达O-A-Asia1型FMDV主要免疫原性基因的重组伪狂犬病毒1株；（3）表达FMDV免疫原性基因的杆状病毒2株；（4）表达猪源IFN-α/IFN-γ和A型FMDV P1基因的重组杆状病毒3株；（5）表达口蹄疫和小反刍兽疫病毒主要保护性抗原基因的重组羊痘病毒2株。成功研制CpG-IFN、CpG-IL4以及多磷腈和CpG DNA等生物复合佐剂3种，纳米乳油佐剂、纳米粒IL-2佐剂以及多孔硅和上转换荧光纳米材料的载药系统等纳米复合佐剂材料4种，以及IL-2、IL-4和IFN-γ等多种哺乳动物细胞表达质粒和多糖类免疫增强剂4种。

关键词：口蹄疫 病毒 新型疫苗 基因工程修饰病毒疫苗株 免疫佐剂

Abstract：More than 10 kinds of the new type vaccines is developing. There are 5 kinds of the new vaccine made in major progress. The first one is bivalent synthetic peptide vaccine of FMD type O and type Asia1， the second one is synthetic peptide vaccine in pig of FMD type O（peptide 2600+2700+2800）， the third one is type A recombinant strains （Re-A/WH/2009） of type O、type A and type Asia1 trivalent inactivated vaccine， the fourth one is the marker vaccine of FMD type O， and the last one is broad-spectrum inactivated virus gene engineering vaccine in pig of FMD type O. The first four vaccines were all safe to the animals， PD50 values were greater than 6.0， the vaccine immune duration is 6 months， the shelf life of synthetic peptide vaccine is 12 months， and all has applied for a new veterinary drug registration. The first one has completed all laboratory studies and clinical trials， and has passed the preliminary examination for new veterinary drug application. The second one has completed all laboratory studies and clinical trials， and has passed the inspection test and review. The third one has got the new veterinary drugs registration certificate， and realized industrialization. The last one has applied clinical trial. Varying degrees of progress has been made in other vaccine research. With the reverse genetics technology platform， five genetically engineered vaccine candidate of FMDV were screened and constructed， which were rV-STN-5， O/rV-1， Re-A/WH/2009， Re-Mya/98/BY/2010 and Re-Asia1/HN/2006. Using gene cloning and recombination technology， other nine genetically engineered vaccine candidate of FMDV were constructed， respectively is：（1） PRV TK-/gE-/PanP12A-P1；（2） PRV TK-/gG-/OAY；（3） Two recombinant baculovirus which expressing the immunogenicity gene of FMDV；（4） Three recombinant baculoviruses which expressing interferons alpha/IFN–gamma of porcine and P1 gene of FMDV type A；（5） Two restructuring goatpox viruses which expressing the major protective antigen gene of FMDV and small ruminants virus. Three biological compound adjuvants containing CpG-IFN， CpG-IL4， multi-phosphonitrile and CpG DNA， four nano composite adjuvants including Nano EC adjuvant， nanoparticle adjuvant IL-2， and drug-loaded system of porous silicon and upconversion fluorescence nanomaterials， four mammalian cell expression plasmids which can expressing IL-2， IL-4 and IFN-γ as well as polysaccharides immunopotentiator have successfully developed.

Key Words：Foot and mouth disease virus；New type vaccine；Genetically engineered vaccine candidate；Immunologic adjuvant

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