【作者】 张晓勇；

【导师】 陈溥言；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2005， 硕士

【摘要】 猪圆环病毒2型（PCV-2）与猪群中发生的多种疾病相关,包括断奶仔猪多系统衰竭综合征（PMWS）、猪皮炎肾病综合征（PDNS）、A2型先天性震颤（CT）等。其中以PMWS最为严重,已经给全世界养猪业带来了很大的经济损失,国内也未能幸免。目前,国内还没有有效的检测试剂盒,快速、特异的检测方法已成为迫切需要。PCV-2的Cap蛋白为病毒的主要结构蛋白,由病毒基因组中ORF2基因编码,构成病毒的核衣壳,具有较强的免疫原性,基因序列保守,可应用于临床诊断。本研究则从以下方面研究Cap蛋白,旨在建立PCV-2检测方法,为诊断与该病毒相关的疾病奠定基础。 1.设计一对引物从谈国蕾构建的pcDNA3-ORF2载体中扩增出ORF2基因,并将扩增出的片段插入毕赤酵母表达载体p PICZ。A中,构建成重组质粒p PICZ_α-ORF2,经甲醇诱导表达、SDS—PAGE、间接ELISA和Western blot试验,结果表明:ORF2基因片段能被分泌表达,且具有良好反应原性。 2.研究了培养条件与表达产量的关系,确定高拷贝重组酵母表达ORF2蛋白的最佳条件为:28-30℃,225rpm振荡培养至BMGY中菌体OD₆₀₀值为3时,将菌体转入pH7.5的BMMY培养基中使菌体浓度为OD值1.5,培养72h,每24h加入甲醇使终浓度达0.5%。在最佳条件下ORF2蛋白的表达量可达278ug/mL,通过比较不同温度下保存ORF2蛋白的降解率得出酵母培养物上清应保存于-20℃。 3.以ORF2蛋白作为包被抗原初步建立了检测猪圆环病毒2型抗体水平的间接ELISA方法,并对该方法的实验步骤进行了优化,优化后的步骤是:42.5μg/mL抗原4℃过夜包被,用1%BSA封闭3h,再与1%BSA稀释的血清作用45min,接着与酶标二抗在37℃作用30min或室温下作用45min,然后用OPD在37℃作用15min或室温下作用20min,最后用0.12%HF终止反应。用此优化方法检测了200份待检猪血清样品。并将其与华中农业大学的圆环病毒病酶联免疫吸附试剂盒方法相比较,检出符合率达到90%,具有广阔的应用前景。更多还原

【Abstract】 Some kinds of diseases are related with porcine circovirus type 2 in porcine groups including PMWS, PDNS, CT, et al. Especially, PMWS is the most serious and has made great economic losses in the world. China can not escape by sheer luck. At present, there is no efficient diagnostic kit in the domestic market. The rapid and specific diagnostic method is much important to control these diseases .The nucleocapsid protein of PCV-2 encoded by ORF2 is a major structural protein of virus and has specific immunogenicity .The Cap gene is a conserved sequence in the viral genome .The expressed product would be used in diagnosis .So as follows in this research are to set up the diagnostic method on the basis of Cap protein and protect against the diseases associated with PCV-2.1. we designed one pairs of primers and amplified the ORF2 genes from the vector pcDNA3-0RF which was constructed by Tan, and the PCR products were inserted into secretory p PICZ. A P. pastor is expression vectors. Recombinant strains with ORF2 gene were acquired and induced with methanol. SDS-PAGE and Western blot analysis showed that the gene could be expressed in P. pastoris and the products had good reacting ability.2. We studied the relations between expression yield and growth conditions and found the optimal expression conditions of the recombinant ORF2 protein were: 28-30 ℃, 225rpm, when BMGY with OD600 being 3, the recombinant Picha Pastoris was add to the BMMY medium of pH 7.5which with OD600 being 1.5.Then the solution was incubated 72h and methanol was added to keep its concentration up to 0.5% between 24h.After induction under optimal conditions, the yield of ORF2 protein could be added up to 278μg/mL. It was found that the supernatant should be conserved in -20 ℃ after comparing effects of different conservation temperature on lysis of protein ORF2.3. An indirect ELISA using protein ORF2 as coating antigen was established to monitor antibodies level of pigs against PCV-2 and its operation methods were optimized. The optimal operation methods were: A protein concentration of 42.5μg/mL was coated overnight at 4℃ on 96-well microtiter plates and the plates were subsequently blocked 3h with 1% BSA ,and then incubated for 45 min with sera diluted by 1% BSA, after washing the plates, the anti-swine immunoglobulin conjuate was added and incubated for 30 min at 37℃ or 45min at RT, and then the indicator OPD was added and incubated for 15 min at 37℃ or 20min at RT, 0.12% HF was added before OD was measured at 630nm.200 pieces of sera from pigs infected by PCV-2 were examined by this method and compared with the method of latex agglutination diagnostic kit which constructed by HuaZhong Agriculturaluniversity. The agreement ratio between the two methods was 90%. Experiments suggested that the method we constructed was promising.更多还原