【作者】 王硕；

【导师】 李俊；

【作者基本信息】 山东师范大学 ， 动物学， 2020， 硕士

【摘要】 猪圆环病毒(Porcine circovirus,PCV)可侵害断奶仔猪和育肥猪并能够引发多种与猪圆环病毒相关的疾病,是当前世界养猪业的巨大威胁,严重影响着养猪业的发展与进步。随着研究发现,病毒的各个血清型对猪体的抗原性、致病性以及基因同源性存在着及其明显的差异,因此人们将猪圆环病毒划分成猪圆环病毒1型(Porcine circovirus 1,PCV1)、猪圆环病毒2型(Porcine circovirus 2,PCV2)以及猪圆环病毒3型(Porcine circovirus 3,PCV3)三种基因型。已知PCV1无致病性,而PCV2有较强的致病性,是造成猪圆环病毒相关性疾病(Porcine circovirus-as sociated diseases,PCVADs)的主要病原,其中危害最严重的是断奶仔猪多系统衰竭综合征(Postweaning multisystemic wasting syndrome,PMWS),该病在我国乃至世界猪群中广泛存在,已成为一种常见病和多发病。PCV3感染可引发猪的皮炎肾病综合征、母猪繁殖障碍等,患病仔猪出现多系统炎症反应,导致器官功能紊乱,严重影响全世界的生猪养殖业的发展。由于PCV2与PCV3的衣壳蛋白氨基酸同源性低至30%,两种病毒间几乎不存在交叉免疫保护。目前,仍没有可同时预防和控制PCV2与PCV3的疫苗和药物。本研究将PCV2与PCV3的Cap蛋白利用杆状病毒表达系统融合表达,不仅有助于PCV2与PCV3的疫苗的研制,还可为研制诊断试剂盒提供蛋白。利用该系统同时表达PCV2 Cap蛋白与PCV3 Cap蛋白,并进行后续纯化及疫苗的研制,比传统方式更加安全有效,为病毒引起的相关疾病的控制和预防PCV的感染具有重要的意义。本研究共分为以下几个方面:2013-2018年山东省猪圆环病毒2型的遗传演化分析:为揭示近年来PCV2在山东省遗传演化情况,分析PCV2代表性优势毒株,为政府防控决策做进一步参考。我们分析了2013年至2018年在山东省上传到GenBank的40个PCV2基因组全序,使用MEGA V5.0软件构建全基因组进化树。系统进化树分析表明,山东省PCV2基因型的演化情况为:3种基因型共存(2a,2b,2d),发现目前PCV2d为主要流行毒株。通过对不同基因型ORF2编码的氨基酸序列分析,发现不同基因型中存在特定的氨基酸位点,主要分布在ORF2编码的81–160位氨基酸之间。PCV2 Cap蛋白与PCV3 Cap蛋白在杆状病毒表达系统中的表达:利用昆虫细胞-杆状病毒表达系统同时表达PCV2Cap和PCV3 Cap,并在重组序列前段加上一段蜂素信号肽序列便于其分泌表达。选用双启动子表达重组杆状病毒,间接免疫荧光结果显示重组杆状病毒能分别与鼠抗PCV2 Cap和鼠抗PCV3 Cap单抗特异性结合;基因组PCR鉴定结果显示,已成功扩增出与相应目的片段大小相符的特异性产物;Western-Blot鉴定可见分子量为70kD左右的蛋白条带;本实验运用杆状病毒表达系统成功表达出PCV2 Cap和PCV3 Cap蛋白,为表达融合蛋白提供新方法。重组杆状病毒的高效表达:将表达条件进行优化以促进重组蛋白高效表达。根据测定的P4代重组杆状病毒滴度结果,并分别以MOI=1,0.5,0.1感染sf9细胞,设置24h、48h、72h、96h、120h五个时间段来收获病毒液。经过实验优化后的融合蛋白获取条件为:MOI=0.1接种2.5×106个/mL的sf9细胞,接种后4天收获融合蛋白,平均表达量可达0.7mg/mL,最高表达量可达0.85mg/mL。该方法能够简单高效的表达PCV2 Cap和PCV3 Cap融合蛋白,是目前其他方法的表达量的2-8倍,极大的增加了表达效率,为进一步研制和生产PCV2、PCV3二联亚单位疫苗建立了良好的理论基础。更多还原

【Abstract】 Porcine circovirus can infect weaned piglets and fattening pigs,and can cause a variety of diseases related to porcine circovirus.It is a huge threat to the pig industry in the world today,which seriously affects the development and progress of pig industry.According to the differences in the antigenicity,pathogenicity,and genetic homology of the serotypes of the virus to pigs,Porcine Circoviruses are classified intoPorcine Circovirus type 1,Porcine Circovirus type 2,and Porcine Circovirus type 3.PCV1 is known to be non-pathogenic,and PCV2 is the main cause of porcine circovirus-as sociated diseases(PCVADs).The most serious of these is Postweaning multisystemic wasting syndrome,(PMWS),which is widespread in China and the world,and has become a common and frequently-occurring disease.PCV3 infection can cause dermatitis nephrotic syndrome in pigs,reproductive disorders in sows,and so on.Multi-system inflammatory reactions occur in diseased piglets,leading to organ dysfunction,which seriously affects the development of pig farming worldwide.However,the amino acid homology of the capsid proteins of PCV2 and PCV3 is as low as 30%,and there is almost no cross-immunity protection between the two viruses.Currently,there are no vaccines and drugs that can prevent and control both PCV2 and PCV3.In this study,the baculovirus expression system was used to express the Cap proteins of PCV2 and PCV3,which will not only help the development of PCV2 and PCV3 vaccines,but also provide proteins for the development of diagnostic kits.Using this system to simultaneously express fusion proteins and perform subsequent purification and vaccine development is safer and more effective than traditional methods,and has important significance for the control of virus-related diseases and prevention of PCV infection.This study is divided into the following areas:Analysis of genetic evolution of Porcine circovirus type 2 in Shandong Province from 2013 to 2018: To reveal the genetic evolution of PCV2 in Shandong Province and analyze representative PCV2 dominant strains,we analyzed 40 cases uploaded to Gen Bank in Shandong Province from 2013 to 2018.Complete PCV2 genome sequence was constructed using MEGA V5.0 software.Phylogenetic tree analysis showed that the evolution of PCV2 genotypes in Shandong Province was the coexistence of 3 genotypes(2a,2b,2d),and PCV2 d was the main epidemic strain.Analysis of the amino acid sequences encoded by ORF2 of different genotypes revealed that specific amino acid sites existed in different genotypes,mainly distributed between amino acids 81-160 encoded by ORF2.Expression of PCV2 Cap protein and PCV3 Cap protein in baculovirus expression system: Insect cell-baculovirus expression system is used to express PCV2 Cap and PCV3 Cap simultaneously,and a section of apicin signal peptide sequence is added to the front of the recombination sequence to facilitate its secretion and expression.Double promoters were used to express the recombinant baculovirus,and indirect immunofluorescence results showed that the recombinant baculovirus can specifically bind to mouse anti-PCV2 Cap and mouse anti-PCV3 Cap monoclonal antibodies,respectively.Genomic PCR identification results showed that it had been successfully amplified and corresponding a specific product with the size of the target fragment;further Western-Blot identified a protein band with a molecular weight of about 70 k D;this experiment successfully expressed a protein containing the PCV2 Cap gene and the PCV3 Cap gene using a baculovirus expression system.Efficient expression provides new methods.Efficient expression of recombinant baculovirus: Optimize the expression conditions to promote efficient expression of recombinant proteins.According to the measured results of P4 recombinant baculovirus titers,sf9 cells were infected with MOI = 1,0.5,and 0.1,respectively,and the virus liquid was harvested by setting five time periods of 24 h,48h,72 h,96h,and 120 h.The experimentally optimized fusion protein acquisition conditions were: MOI = 0.1 to inoculate 2.5 × 106 cells/m L of sf9 cells,and the fusion protein was harvested 4 days after inoculation,with an average expression of 0.7 mg/m L and a maximum expression of 0.85 mg/m L.This method can express PCV2 Cap and PCV3 Cap fusion protein simply and efficiently,which is2-8 times the expression amount of other methods at present,which greatly increases the expression efficiency.It is established for the further development and production of PCV2 and PCV3 duplex subunit vaccine a good theoretical foundation.更多还原