【作者】 秦春香；

【导师】 谢芝勋；

【作者基本信息】 广西大学 ， 预防兽医学， 2007， 硕士

【摘要】 根据GenBank上的禽呼肠病毒（ARV）S1基因序列，设计并合成了一对跨越P10和P17非结构蛋白基因的特异性引物，对13个ARV毒株进行RT-PCR扩增、克隆及序列测定。结果显示，13个ARV毒株的P10蛋白基因ORF全长均为297bp，编码98个氨基酸；P17蛋白基因ORF全长为441bp，编码146个氨基酸。这13个ARV毒株P10、P17蛋白基因核苷酸同源性分别在96.6％～100％和98.2％～100％之间，推导的氨基酸同源性分别在91.9％～99.0％和95.2％～99.3％之间。将这13个ARV毒株与GenBank上其他正呼肠病毒毒株，包括番鸭株（DRV）和飞狐上分离的内尔森海湾病毒（NelsonBayvirus，NBV）及两个澳洲分离株（ARM21和SOM24）进行同源性比较和遗传进化树分析，结果表明，呼肠病毒有地域和种类的差别。对ARV S1133株的P17非结构蛋白基因完整ORF进行RT-PCR扩增、与PGEX-4T-I原核表达载体连接，转化大肠杆菌BL21感受态细胞，经0.2mmol／L的异丙基硫代-β-D-半乳糖（IPTG）诱导表达，SDS-PAGE电泳分析表明，表达的融合蛋白分子量为42.4kD，占全菌蛋白的34％。Western-blotting结果表明：原核表达的P17重组蛋白具有抗原性，能与活病毒感染的ARV阳性血清反应。另外，诱导表达了ARV的两个结构蛋白δ₃和δ₂以及两个非结构蛋白δNS和P17，分别用不同浓度的尿素和Triton X-100对这四个蛋白进行包涵体纯化，各蛋白的终浓度分别为：38mg／mL、48mg／mL、28mg／mL、46mg／mL。经BandScan4.3生物图象分析软件分析各蛋白的纯度分别是：68.6％、75.7％、84.0％和85.0％。达到较高的浓度和纯度，说明本试验探索的纯化方法能有效的纯化这些重组蛋白。最后，将纯化的δ₃、δ₂和δNS、P17蛋白分别作为包被用抗原进行间接酶联免疫吸附试验（ELISA），确定其最佳包被浓度分别为9.5μg／mL、12.0μg／mL和9.3μg／mL、11.5μg／mL；一抗稀释度均为1∶200；HRP酶标羊抗鸡IgG稀释度为1∶5000，四种重组蛋白均不与NDV、AI和IBDV阳性血清发生交叉反应，具有较好的特异性。本研究初步建立了能检测ARV抗体的δ₃-ELISA、δ₂-ELISA方法，以及δ₃、δ₂两个蛋白同时包被的δ₃-δ₂ELISA方法。用这三种方法对ARV活病毒感染和灭活疫苗免疫鸡的血清进行检测，表明δ₃-δ₂-ELISA方法在ARV抗体检测方面具有较高的敏感性。用相同的方法建立了能鉴别ARV灭活疫苗免疫和活病毒感染的δNS-ELISA和P17-ELISA方法，以及δNS、P17两个蛋白同时包被的δNS-P17-ELISA方法，并对这三种方法进行敏感性比较及统计学分析，结果表明两个非结构蛋白同时包被的ELISA方法比单个抗原包被的方法能更好的区分ARV灭活疫苗免疫与活病毒感染。更多还原

【Abstract】 A pair of specific primers were designed and synthesized according to S1 genesequences of Avian reovirus（ARV） from GenBank, which included the whole open readingframe（ORF） of P10 and P17 nonstructral protein genes.Both P10 and P17 protein genes of 13ARV strains were amplified,cloned and sequenced.The whole ORF of P10 and P17 proteingenes of those 13 ARV strains were 297bp and 441bp in length,respectively.And they werepredicted to encode P10 and P17 nonstructral proteins of 98 and 146 amino acids,respectively.The percent ages of nucleotide and aminoacid sequence identities weredetermined.The nucleotide and aminoacid sequence identities of P10 protein gene amongthose 13 ARV strains exhibited 96.6％-100％and 91.9％-99.0％,respectively.The identities ofP17 protein gene showed 98.2％-100％and 95.2％-99.3％, respectively. Comparison of thenucleotide and predicted aminoacid sequences and phylogenetic analysis between ARV andother orthoreoviruses （DRV, NBV, RAM21,SOM24） showed that Reovirus had the divergenceamong regions and species.RT-PCR amplify the Whole Reading Frame （ORF） of P17 protein gene from ARV S1133strains. The product ligated with PGEX-4T-1 Prokaryotic expression Vector, and transformedinto Escherichia BL21 competent cell. The recombination was induced with 0.2mmol/L ofIPTG. The results of SDS-PAGE analysis revealed that the molecular weight of the expressedproduct was 42.4kD. Western-blotting analysis showed that the Prokaryotic expression P17recombinant protein could be specifically recognized by the positive sera of avian infectedwith ARV.In additional,two structural proteinδ3 andδ2,and two nonstructural proteinδNS andP17 of ARV were Prokaryotic expressed.The inclusion bodies of four proteins were purifiedby urea and Triton X-100.The concentration of these four proteins were 38mg/mL、48mg/mL、28mg/mL、46mg/mL, respectively. The purity of urea-purified fusion proteins were 68.6％、 75.7％、84.0％and 85.0％, respectively. Achieves a higher density and purity, which showedthat this experimental exploration can effective purify the four recombinant proteins.Finally, These four proteins were used for coating antigen of indirect enzyme-linkedimmunosorbent assay （ELISA） after purified.The optimal conditions was determined.It wasshown that the optimal of the coating concentration of these proteins were 9.5μg/mL, 12.0μg/mL,9.3μg/mL and 11.5μg/mL respectively, all the optimal dilution of serum sample were1:200, the optimal dilution of enzyme-labeled goat-chicken IgG were 1:5000. These fourrecombinant proteins showed no reaction with antiserum to NDV, AI,IBDV.Which indicated that these indirect ELISA had good specifity for detetion of ARVantibody in serum. Our research had developmented ofδ₃-ELISA,δ₂-ELISA andδ₃-δ₂ELISAmethods,and use these methods to detected the serums which were infected with ARV orinjected with died vaccine.The result showed thatδ₃-δ₂-ELISA was the most sensitivity inARV antibody detection.We used the same method to development ofδNS-ELISA,P17-ELISA andδNS-P17-ELISA, It show that detection of antibodies of ARVnonstructural proteinδNS and P17 can distinguish between vaccinated and infected chickens.Compared to the sensitivity ofδNS-ELISA,P17-ELISA andδNS-P17-ELISA,and relationwith statistics analysis,we can indicated that theδNS-P17-ELISA which two nonstructuralproteins as coating antigen simultaneity has advantages over theδNS-ELISA and P17-ELISAin distinguish between ARV vaccinated and infected serum samples.更多还原