【作者】 徐敬龙；

【导师】 胡仲明； 金宁一；

【作者基本信息】 吉林大学 ， 基础兽医学， 2005， 硕士

【摘要】 将本室构建和筛选的共表达NDV F、IBDV VP0 基因重组鸡痘病毒(重组鸡痘病毒vFV282)经不同的理化因素处理,接种到鸡胚成纤维细胞(CEF)上,观察处理前后重组鸡痘病毒vFV282 对CEF 的毒力和感染滴度,获得该病毒的部分理化学特性。通过鸡胚绒毛尿囊膜传代、CEF 传代和鸡体传代,检测回收毒的毒力、感染滴度,通过PCR 方法检测F、VP0 基因。本试验通过测定不同免疫途径和方法(一次刺种;刺种首免;首免后28d 二免;肌肉注射)和不同时间采血测抗FPV、NDV、IBDV 的中和抗体效价和ConA 刺激脾淋巴细胞反应的净增值,来分析重组鸡痘病毒vFV282 体液免疫和细胞免疫的消长规律和水平。以上结果为研究重组鸡痘病毒vFV282 免疫水平与免疫攻毒效力的平行关系,为建立效力检验方法和标准,为研究免疫期等奠定了坚实的基础。更多还原

【Abstract】 Examination of resistance against different physicochemical agents isthe important content of complete identification of a virus. Physicochemicalagents can influence the living and characteristic of virus, even caninactivate virus.Various kinds of physicochemical agents, such as heat, radiation, pHand chemical inactivator, can change and destruct viral nucleic acids, whichhinder replication, transcription and translation of virus, consequencely.Some physicochemical agents can also influence or destroy capsids orenvelopes of virus, which result in loss of viral infectivity too. Further more,many physicochemical agents have the ability of change and destruct viralnucleic acids and proteins simultaneously.In summary, it is very significance to understand the effect of variousphysicochemical agents in inactivation of virus. Because various viruseshave different sensitivity to various physicochemical agents, it is veryimportant to keep these agents far away during the preservation of virus.Belonging to genetic engineering recombinant virus, recombinantFowlpox virus (rFPV) vaccine vFV282 strain has favourableimmunogenicity, which co-express VP0 gene of Infectious Bursal DiseaseVirus (IBDV) and F gene of Newcastle Disease Virus (NDV), can preventeffectively Fowlpox, Newcastle Disease (ND) and Infectious BursalDisease (IBD) in one needle. So it significance to understand someimportant physicochemical characteristic for large scale production,lyophilization, preservation and sterilization of rFPV vaccine vFV282.In this study, after treatment by various physicochemicalagents, including temperature, HCl (pH3), NaOH (pH9), ether,chloroform, and trypsogen, rFPV vFV282 was inoculated in CEF.And the changes of virulence and infectivity of vFV282 wereevaluated by examined TCID50, in order to analyze the influence of theseagents on vFV282. The results indicated that the change of pH(3.0~9.0) didn’t lead to apparent change of virulence and infectivity.However, vFV282 could be inactivated by 55℃for 60min or 60℃for 15min. vFV282 was sensitive to chloroform, so that infectivitydropped 5.5 log10TCID50 after treatment with 5% chloroform for 10 min.Treated with 1% trypsogen in 37℃for an hour, TCID50 reduced 2.55log10TCID50. These results indicated the susceptiveness of vFV282towards trypsogen. Results also suggested the resistance of vFV282towards ether, for infectivity reduced only 1.0 log10TCID50 after treatmentwith ether for 24 hours.In order to evaluate the inherent stability of vFV282, we examined theF gene of NDV and VP0 gene of IBDV carried by vFV282. First, vFV282was cultivated on SPF embryoes of chicken 10 passages continuously. Then,passage 3rd, 6th, 10th were identified by PCR and examination on TCID50of vFV282. Secondly, vFV282 was cultivated on CEF 30 passagescontinuously. And passage 1st, 5th, 10th, 15th, 20th, 25th, 30th wereidentified. Finally, vFV282 was cultivated 8 passages on chickenscontinuously, and 3rd, 5th, 8th were identified. The results indicated thatlost of VP0、F gene did not happen, and TCID50 did not change.Furthermore, VP0 gene and F gene were cloned into pMD18-T vector andsequenced, respectively. The result confirmed the stability of these twogenes.Previous studies proved rFPV vFV282 could replicated in chickens,expressed and presented F gene of NDV, VP0 gene of IBDV and wholegenes of FPV, and induced the specific antibodies, which could enhancecellular immunity of chicken. In this study, chickens were inoculated usingvFV282 by various vaccination strategies. And neutralizing antibodiesagainst FPV, NDV, IBDV and lymphoproliferation of vaccinated chickenwere examined respectively, in order to analyze the profile and intensity ofspecific humoral and cellular immune responses.Chickens of 7-days old were divided into 4 groups, such as controlgroup, intramuscular injection group, intradermal injection once group,intradermal injection twice group. Experimental groups inoculated a dozeof vFV282 (≥105.5TCID50), and control group was inoculated saline.Post-vaccinated, blood and spleen of chickens were collected on 7d, 14d,21d, 28d, 35d, 42d, 49d and 56d. Specific neutralizing antibodies anti-FPV,anti-NDV and anti-IBDV were detected respectively. And Tlymphoproliferations were also examined by MTT assay.The results showed that the specific neutralizing antibodies ofchickens belonging to intradermal injection group and intramuscularinjection group rose rapidly from 7dpv, arrived peak on 14~21dpv, anddropped from 28dpv, but still kept a favourable level on 42dpv. Inintradermal injection group, anti-FPV neutralizing antibody rose veryquickly from 7dpv, arrived top level on 14dpv (1:44.67), dropped from28dpv (1:17.70), still kept calm on 42dpv (1:9.25); anti-NDV neutralizingantibody rose directly from 7dpv, peaked on 14dpv (1:89.13), descendedfrom 28dpv (1:19.63), still had a weak extent (1:14.19); anti-IBDVantibody rocketed from 7dpv, reached top level on 21dpv (1:89.13),更多还原