【作者】 王永山；

【导师】 陆承平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2004， 博士

【摘要】 兔出血症(Rabbit Hemorrhagic Disease，RHD)是由兔出血症病毒(Rabbit Hemorrhagic Disease Virus，RHDV)所致的兔的一种急性、败血性、高度致死性传染病。该病于1984年发现，其后在世界其它地区相继发生。迄今，国内外各养兔地区仍有存在或流行，是养兔业的严重威胁。 本研究用分子克隆技术从具有良好免疫原性的RHDV中国早期流行毒株NJ85中成功克隆病毒衣壳蛋白(VP60)全长基因，序列测定该基因长度为1740 nt，编码579aa，并在GenBank注册(GenBank Accession AY269825)。运用GenBank数据库、生物信息学和结构生物学方法，全面系统地分析了17年来世界各地RHDV毒株VP60基因的遗传变异，NJ85与WX84、TP二个中国毒株的VP60基因DNA序列的同源性分别为92.7％和97.2％，氨基酸序列的同源性分别为96.1％和98.6％，与其他国家16个毒株的VP60基因DNA序列的同源性在83.7％～97.0％之间，氨基酸序列的同源性在90.5％～99.0％之间，高度同源。进一步分析VP60基因的六个分区，A、B、D、F四个区变异率较低，C、E二个区变异率较高，表明A、B、D、F四个区较C、E二个区在病毒衣壳中执行着更多的生物学功能。在遗传进化上，历年来世界各地的RHDV毒株在氨基酸水平上可分为三个支谱系(群)，在核苷酸水平上趋向四个支谱系，谱系没有呈现地理或时间特征。三个中国毒株分布在二个不同的支谱系中。与RHDV同为兔病毒属的欧洲野兔综合征病毒(EBHSV)组成了另一个谱系。以上结果对认识RHDV的变异趋势具有参考价值。分析了NJ85 VP60蛋白的分子量、等电点、疏水性、亲水性、抗原性、表面区、电荷、柔性区、二级和三级(立体)结构等分子结构特征。VP60的二级结构以β片层为主，是形成稳定三级结构的基础。病毒衣壳由90个VP60二聚体组成，二聚体由VP60单体以A/B5和C/C2两种方式构成，单体在二聚体中的构象有A、B、C三种形式，这些蛋白质分子形成拱形双体结构，从而在表面形成32个杯状凹陷。VP60单体含有S、P两个结构域，两者通过柔性绞链连接，P结构域由VP60的C端部分形成，P分为P1和P2二个亚结构域，P2位于病毒衣壳表面，含有病毒株特异性抗原表位和红细胞结合位点。本研究为RHDV的分子生物学增添了新的资料。兔出血症病毒中国早期流行毒株NJ85衣壳蛋白基因的分子结构特征及其表达应用 原核表达系统具有表达量大、周期短以及成本低等优点.本研究从免疫效果、生产成本和产业化要求出发，选用原核表达系统，成功构建了NJ85毒株VP60基因高效原核表达质粒，用SDS一PAGE和凝胶扫描分析，重组菌VP6O蛋白的诱导表达量为40%，分子量约62 kD，在菌体内以包涵体形式存在，免疫印迹试验证明重组VP60蛋白具有杭原性。此前，国内外虽已克隆表达了多个RHDV毒株的VP60基因，但表达量在21%以内 .NJ85VP6O基因的高效表达为研制具有自主知识产权且经济实用的RHD新型检测试剂和基因工程疫苗莫定了基础. RHDV抗体的检测目前多采用血凝抑制试验(HI)，该方法在每次试验前都要进行杭原标定、同时要制备红细胞和对血清样本处理，不仅繁琐而且结果不稳定，重复性差.ELISA具有灵敏度高、特异性强和稳定性好的优点，但是，由于RHDV迄今未能稳定地适应于任何细胞培养，从RHD病理组织中获得大量的高纯度的VP60杭原，成本较高，本研究用重组VP6O解决了这一问题. 以重组VP60蛋白为抗原，建立了RHDV抗体间接ELISA检测方法.优化的试验反应条件为:重组vP60的包被浓度为1 .0林留mL，用10%牛血清封闭，以大肠杆菌提取物稀释被检兔血清.进一步研制成功R壬IDv抗体间接ELISA检测试剂盒，测定了其主要指标，制定了各成分的质量控制标准.对各种不同免疫状态兔血清中的Rl硕DV EUSA与Hl抗体的相关性进行了研究，二者呈正相关.分析11个RHD免疫兔场的1 130份血清样本，各免疫兔群血清中的RHDv抗体水平存在差异，成5。值在1 .09一1 .76之间，显著高于非免疫兔血清(0 .05)和SPF兔血清(0 .02)，低于高免兔血清(2.34).以大肠杆菌表达的蛋白作杭原用于检测动物体内的抗体，面临的一个问题是纯化的抗原中存在大肠杆菌成份的污染而使检测结果出现假阳性，用大肠杆菌提取物稀释被检血清解决了这一问题.本研究制备的试剂盒为兔群免疫学监测和评价疫苗的免疫效果提供了便利。 为了验证重组VP60对兔的免疫保护效果，将34只清洁兔随机分成3组:单用免疫佐剂对照组(8只)、单用重组VP60组(8只)和重组VP6O加免疫佐剂组18只)，分别经皮下注射，vP60的免疫剂量为400林留只/次，免疫2次，间隔7d。在第2次免疫7d后，VP6O加免疫佐剂组的兔血清杭体水平显著高于单用VP6O免疫组(ELISAl:64000对比l:4000/Hll:1 28对比l:16)，表明免疫佐剂可明显加速重组vP60诱导抗体产生的进程。此时，用RHDV强毒攻击，免疫佐剂对照组与单用VP60免疫组的实验兔均全部死亡(8/8和8/8)，VP60加免疫佐剂组则全部存活，保护率为100%中文摘要(1 8/18).用RHDV血凝试验(HA)检测，死亡兔的肝脏组织液HA效价大于1:512，而存活兔的肝脏组织液HA效价小于l:2，肝脏组织触(涂)片检查和细菌培养均为阴性，证?更多还原

【Abstract】 Rabbit hemorrhagic disease (RHD) caused by Rabbit hemorrhagic disease virus (RHDV) is characterized by an acute course, hemorrhagic septicema and a high mortality in adult rabbits whereas infected rabbits below the age of about 2 months usually survive. It was first reported in China in 1984 and occurred later in other countries successively. Today, RHD is widely distributed around the world and seriously threaten the farming of rabbits.In our study the capsid protein (VP60) gene of the Chinese early isolate NJ85 of RHDV was amplified by reverse transcribed polymerase chain reaction (RT-PCR), cloned and sequenced. VP60 gene of NJ85 was in size of 1740nt and encoding 579aa. Comparison with other Chinese field isolates WX84 and TP showed that the homology were 92.7% and 97.2% for nucleotide sequence; 96.1% and 98.6% for amino acid sequence respectively. Alignment with other 16 sequences of RHDV isolated from other countries registered in GenBank exhibited that the homology was 83.7%~97.0% for nucleotide and 90.5%~ 99.0% for amino acid sequence. The results indicated that the sequence of VP60 in different RHDV isolates was highly homologous. In the all six regions of VP60, low degree of variation of VP60 was found in the region A, B, D, F and relative high degree of variation in the region C and E. Based on VP60 sequence, phylogenetic analysis showed that the historic RHDV isolates were divided into 3 branches of the lineage of RHDV according to the amino acid sequences and 4 branches according to the nucleotide sequences. There was no obvious correlation between geographical region, historic period and homological degree. Three Chinese field isolates lay in 2 different branches of the RHDV lineage, in the meanwhile EBHSV formed another lineage. The molecular weight, isoelectric point, hydrophobicity, 2-dimensional and 3-dimensional structure of NJ85 VP60 were determined and predicted on the theory of bioinformatics. The major secondary structure, B-sheet contributed to the stability of VP60. Based on the structural model, the architecture of NJ85 capsid was 32 cup-shaped depressions. The capsid was composed of 90 A/B5 and C/C2 dimers formed by the single unit of VP60. The conformation of thesingle unit in dimer existed three forms of A, B and C. The single unit of VP60 had a protruding (P) domain connected by a flexible hinge to a shell (S) domain. P domain was subdivided into two subdomains, P1 and P2. P2 subdomain that located the surface of the capsid contained the determinants of strain specificity and erythrocyte binding site.Prokaryotic expression vectors are usually advantageous to yield the product of foreign purpose gene in large quantities, timesaving and low costs. For this purpose, the full-length of VP60 gene cloned from isolate NJ85 was subcloned into the high-level expression vector pET28. The expression plasmid pET-VP60 was constructed and highly expressed under the induction with IPTG. The proportion of the recombinant VP60 protein reached to 40 percent of the total bacterial proteins under the optimized inductive condition. SDS-PAGE analysis showed that the molecular weight of the recombinant VP60 protein was approximate to 62 kD, and it formed inclusion body in E.coli. The specific reaction of the recombinant VP60 protein with antiserum of RHDV was confirmed by immunoblotting assay. However, The earlier studies on the expression of VP60 the yield of recombinant VP60 was below 21%.Until now the hemagglutination inhibition (HI) test was the popular method for the detection of antibody against RHDV. But it had many inconveniences and disadvantages in practice. In order to overcome the questions, an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibody against RHDV was developed using the recombinant VP60 expressed highly in E.coli as antigen. The optimized conditions for the assay were that the wells of ELISA plate were coated with recombinant VP60 (1.0ug/mL), 10% normal bovine serum was added as blocking agent, and the serum samples were preincubated wi更多还原