【作者】 刘芳；

【导师】 范红结； 王继春；

【作者基本信息】 南京农业大学 ， 兽医硕士（专业学位）， 2016， 硕士

【摘要】 H5N1亚型禽流感(Avian influenza H5N1 subtype,AI H5N1)是当前威胁家禽养殖业安全的重大传染性疾病之一,引起全世界的广泛关注。灭活苗的使用一直是防控禽流感的主要手段,但灭活苗免疫效果差,制备过程中存在散毒风险。基因工程活载体疫苗的研制是新型兽用疫苗研究的热点,在预防疾病流行中具有独特优势。血凝素(HA)是禽流感病毒的主要保护性抗原,可以诱导机体产生中和抗体,是研制禽流感基因工程病毒活载体疫苗的首选抗原性基因。商品化火鸡疱疹病毒载体已成为研制禽用病毒活载体疫苗的良好载体,免疫效果显著优于传统疫苗。疱疹病毒的细菌人工染色体是研制和改进疱疹病毒载体活疫苗的有力工具。本研究应用火鸡疱疹病毒细菌人工染色体克隆,通过基因重组技术将HA表达盒插入到火鸡疱疹病毒基因组的非必需区,成功构建了表达禽流感HA基因重组火鸡疱疹病毒HVTBAC-H5,为禽流感基因工程病毒活载体疫苗的研制打下了良好基础。本研究首先根据公开发表的H5N1亚型禽流感病毒2.3.2.1谱系的HA基因序列,人工合成HA基因,构建了含有该基因和CMV启动子以及poly A终止子的HA基因表达盒。采用一对含插入位点上、下游同源臂的引物,通过PCR方法扩增HA基因表达盒;采用EN PASSANT方法,第一步重组将HA基因表达盒导入火鸡疱疹病毒细菌人工染色体(BACHVT-G)UL54和UL55之间的非编码区。通过PCR和序列测定对重组BAC(BACHVT-HAKAN+)进行鉴定,确认正确后再进行第二次基因重组敲除KAN抗性基因,得到HA重组HVT BAC(BACHVT-G-H5)。通过PCR以及RFLP方法验证所获得的BACHVT-G-H5,结果表明与与预测结果一致。提取BACHvT-G-H5的DNA,应用磷酸钙沉淀法转染鸡胚成纤维细胞(CEF)进行病毒拯救,在紫外光激发下观察病毒蚀斑,结果获得了发出绿色荧光的病毒蚀斑,表明成功拯救出含BAC质粒序列的HVT重组病毒HVTBAC-H5。提取重组病毒HVTBAC-H5基因组DNA,应用一对特异性引物扩增HA基因表达盒并测序鉴定,结果表明HA基因表达盒成功插入,且序列与设计完全一致。通过血凝试验和Western Blotting对重组毒HVTBAC-H5进行鉴定,结果表明插入的外源基因HA能够正确表达,且可与H5N1阳性血清特异性结合,具有抗原性。本研究成功构建了表达禽流感HA重组火鸡疱疹病毒,HA基因在火鸡疱疹病毒基因组内正确插入,且没有发生变异,该研究表明应用HVT BAC(BACHVT-G),通过EN PASSANT 方法能够高效地构建HVT重组病毒,为研制新型禽流感病毒HA重组火鸡疱疹病毒活载体疫苗奠定了基拙。更多还原

【Abstract】 H5N1 avian influenza(H5N1 AI)has become a serious threat to the poultry industry,and received extensive concerns worldwide.Inactivated vaccine has been the primary method of prevention and control of the disease,but can provide poor protection against the pathogen.Furthermore,there might be a risk for live virus transmission during the preparation of the vaccine.The development of genetically engineered live vector vaccine has recently become a research hot spot avian influenza due to its unique advantages in the prevention of infectious diseases.HA gene is therefore the preferred antigen gene in the development of genetically engineered vaccine against avian influenza.Commercialization of turkey herpes virus(HVT)vector has become a good vector for live vaccines against poultry viruses HVT vector has higher immune efficacy compared with conventional vaccines..The herpesvirus bacterial artificial chromosome(BAC)has become a powerful method for the development and improvement of recombinant herpesvirus vaccines.In this study,a recombinant HVT expressing HA of a highly pathogenic AIV(H5N1),named HVTBAC-H5 was constructed through the insertion of HA expression cassette into a non-essential region of HVT genome.The current study has provided a basis for future development of genetically engineered live vector vaccine against AIV.In this study,an HA gene was designed and synthesized based on the sequence data of H5N1 2.3.2.1 published in Pubmed,and an HA expression cassette was constructed containing the HA gene,CMV promoter and poly A terminator.The HA expression cassette was amplified by PCR using a pair of primers containing the homology arms bordering the insertion sites in the HVT genome,and inserted into the non-coding region between UL54 and UL55 on the BACHVT-G by the EN PASSANT method.The obtained recombinant BAC(BAC HVT-HA KAN+)was confirmed by PCR and sequencing,and subjected to a second recombination to knock-out the Kan resistance gene.The obtained virus vector,named BACHVT-G-H5,was confirmed by PCR and RFLP.The BACHVT-G-H5 DNA was extracted,with PCR fragments containing the homology arms and transfected into chicken embryo fibroblasts(CEF)by the calcium phosphate-based method in order to rescue the viros.The recombinant virus HVTBAC-H5 containing the BAC sequence was successfully rescued as indicated by the green fluorescent plaques under UV light.The genomic DNA of HTVTBAC-H5was extracted,and HA expression cassette was amplified and sequenced.It was shown that the designed HA expression cassette had been successfully inserted.Hemagglutinin test and western blot showed that the HA was properly expressed and specifically bound to the H5N1 positive serum.In summary,this study has successfully constructed a HA recombinant HVT.The current method has developed an efficient method for the construction of HVT recombinant virus based on BACHVT-G and EN PASSANT,which provides a basis for the development of novel HVT-vectored vaccine against AIV.更多还原