【作者】 许小琴；

【导师】 韦旭斌；

【作者基本信息】 吉林大学 ， 临床兽医学， 2005， 博士

【摘要】 探索开发防治鸡传染性法氏囊病的新型基因工程疫苗及其免疫佐剂具有重要的临床意义。本课题通过构建传染性法氏囊病病毒JS 株VP2基因真核表达载体及重组杆状病毒表达系统,对VP2 基因疫苗及其Rg1-CpG 佐剂免疫增强作用和作用机理、VP2 基因免疫监测方法等进行了深入研究。结果:构建了JS 株VP2 真核表达载体质粒疫苗及其Rg1-CpG 佐剂疫苗,经SPF 鸡免疫试验和超强毒株的攻毒试验证实,Rg1 100μg+CpG50μg+pcDNA-VP2 100μg/羽组成的佐剂疫苗,经二次免疫,可诱导机体产生与中等毒力活疫苗免疫相一致的抗体效价和抵抗IBDV超强毒株攻击,其保护率高达91.7%;成功构建了重组杆状病毒表达系统,利用该表达系统表达的VP2 蛋白,建立了IBDV 血清抗体的ELISA 检测方法,经稳定性试验证实,该方法对VP2 基因疫苗免疫血清抗体的监测特异性好,灵敏度高,变异度小于4%;并经体外细胞实验研究证实,Rg1-CpG 佐剂具有提高淋巴细胞活性、促进淋巴细胞转化与增殖、诱生干扰素等淋巴细胞因子的作用,表明Rg1-CpG 佐剂增强免疫作用的机制,与激活免疫系统提高综合免疫保护等因素有关。更多还原

【Abstract】 Avian infectious bursal disease (IBD), caused by infectious bursal disease virus(IBDV), is an acute, highly contagious disease. The disease can not only result in suddendeath of chickens, but also cause immunodepression in infected chickens. It was oncesuccessfully controlled by the widespread application of serotype I vaccine. Due to thepresence of variation strains and very virulent strains of IBDV, however, the conventionalvaccines of IBD were no longer so effective than ever before after the half past years of1980’s. Therefore, new vaccines such as gene vaccines and recombinant live virusvaccines become the hot fields in the studies of IBD vaccines. Although some vaccinesbased on the eukaryotic systems have been studied, its low protection rate in immunizedchickens became a big obstacle in application. In our studies, we chose the very virulentIBDV JS strain as experimental materials, successfully constructed the eukaryoticexpression vector as gene immunization of IBDV by cloning VP2 gene into pcDNA3.1,and then studied Rg1-CpG as the adjuvant for immunization of chickens. Moreover, therecombinant VP2 protein of JS strain was successfully expressed in Bac-to-Bacbaculovirus expression systems, and used to establish indirect ELISA to evaluate theefficiency of gene immunization described above.1. Construct of eukaryotic vector expressing VP2 gene of IBDV JS strain and itsbiological characterizationTwenty-four-day-old chickens were intramuscularly injected with the supernatant of100-fold dilution containing IBDV JS strain. The inoculated chickens began to showclassical clinical symptoms of IBD at 48 hours post injection, and die at 120 hours postinoculation. The 1.37 kb fragment of VP2 gene of JS strain was successfully amplified byreverse transcription-polymerse chain reaction (RT-PCR) with primers disigned accordingto sequence of vvIBDV published in GenBank. Sequence analysis of VP2 gene showedthat the large and small hydrophilic regions and seven-peptide region in its deducedamino acid sequence were same as those of standard very virulent strains HK46 (ChineseHongkong), OKYM (Japan), and UK661(British), which implied that JS strain would beof the similar virulence to the very virulent strain. Then VP2 gene was further subclonedinto pcDNA3.1 eukaryotic expression plasmid vector. The recombinant plasmid wasidentified by restriction enzyme digestion. Two fragments, 5.0kb and 1.37kb, were gainedfrom enzyme digestion of BamHI and XbaI, which indicated that the recombinantpcDNA-VP2 was successfully constructed. Transient expression of pcDNA-VP2 wascharacterized in COS-1 cells by indirect immunofluorescent straining with monoclonalantibody to IBDV. The results showed that the VP2 protein was successfully expressed inCOS-1. Chickens (14-day-old) were intramuscularly injected with pcDNA-VP2 plasmidDNA and challenged with standard very virulent strain of IBDV. The results indicated that67% of chickens after second immunization could be protected.2 Expression of VP2 gene of IBDV JS strain in Sf9 cells and its applicationThe VP2 gene of IBDV JS strain was cloned and inserted into the baculovirus transfervector pFastBacI to produce pFastBacI-VP2. DH10Bac competent cells containing thebaculovirus shuttle vector Bacmid were transformed with pFastBacI-VP2 to makeBacmid-VP2. Sf9 cells were transfected by recombinant Bacmid-VP2 DNA andrecombinant virus containing VP2 gene was obtained. The Sf9 cells infected by therecombinant baculovirus with the dosage 5 MOI stop division at 24 hours post-infection.The infected cells and their nuclei increased 1-3 times as normal cells at 48 hours afterinfection. The cells began desquamation at 72 hours, and showed vacuole pathologicalchanges at 96 hours. The expression of VP2 protein in Sf9 cells infected by recombinantbaculovirus was confirmed by indirect immunofluorescent assay, SDS-PAGE andWestern-blotting. The results revealed that the expression product had a molecular weightof 41kD, and could be specifically recognized by multiclonal antibody against IBDV ormonoclonal antibodies. The recombinant VP2 protein expressed in Sf9 cells was used toestablish indirect ELISA for detection of the antibodies to VP2 protein of IBDV.Compared with ELISA established by monoclonal antibody, the established indirectELISA showed high sensitivity and specificity, with 92.3% coincidence. Moreover, thisdetection displayed less 4% variation through the detection of sera collected fromchickens immunized with VP2 gene of IBDV, displaying better stability and reliability. 3 Immune enhancement of Rg1-CpG in immunization of IBDV VP2 gene The LD50 of IBDV JS strain was titered by Karber method in 15 SPF Laihangchickens at 35-day old. The results showed that LD50 of JS strain was 10-3.6, indicatingthat JS strain be of very virulence. The genomic DNA of E.coli strain 037 was prepared for CpG adjuvant by modifidmethods of isolation of DNA. Rg1 of panax and bacterial CpG were used as adjuvant ofgene immunization of pcDNA-VP2, respectively. And 120 SPF chickens were receivedthe mixture containing adjuvant and DNA vaccine. The immunosorbent assay (ELISA)antibody titers against IBDV were detected at 14 days after the first immunization, 7 daysand 14 days after the second immunization, and then the challenge test were preformed inimmunized chickens. The results showed that the antibodies against IBDV in sera ofgroups immunized with the mixture of 100μg of Rg1, 50 μg of CpG and 100μg ofpcDNA-VP2 or group with 200μg of Rg1, 50μg of CpG and 100μg of pcDNA-VP2 couldreach the same levels as the group immunized by middle virulent strain of IBDV. Thedifference of antibody titers between these groups was not significant by the analysis ofSPSS software. However there were significant difference among groups immunized withCpG + pcDNA-VP2, Rg1 + pcDNA-VP2 and plasmid pcDNA3.1. The results alsoindicated that 100μg of Rg1 and mixture of 200μg of Rg1 and 50μg of CpG couldsignificantly enhance immune response. The immune protection rate of the two groupsimmunized with Rg1 + CpG + pcDNA-VP2 reached 91.7%. It was similar to that of thegroup immunized with middle virulent strain, and higher than 58.3% in the groupimmunized with plasmid alone, indicating that the Rg1-CpG should be a very potentialadjuvant for gene vaccines of IBDV. 4 Mechanism of immune enhancement of the adjuvant Rg1-CpG The activities of PMCS lymphocytes in blood collected from chickens immunizedby constructed gene vaccines were assayed by MTT method in vitro. The results showedthat the adjuvant Rg1 and Rg1-CpG significantly improve the activity of lymphocytes. Thevalues of OD570 were 1.012 and 1.052, respectively, with a significant difference ascompared with control group. The transformation rates of lymphocytes of chickensreceiving Rg1-CpG adjuvant and Rg1 adjuvant reached 93.5% and 84.2%, respectively,which was higher than that of control group. The proliferation rate of lymphocytes wasassayed by flow cytometer. The results showed that there were significant differencebetween groups with adjuvant Rg1-CpG and control group, with proliferation rates of22.47% and 4.88%, respectively, indicating the adjuvant Rg1-CpG be of strongerimprovement to the transformation and proliferation of lymphocytes. The detection resultsof induced IFN and other lymphocyte factors indicated that the Rg1-CpG group and Rg1group had higher titers than control group, with significant difference. In the present studies, chickens immunized by constructed eukaryotic expressionvector containing VP2 gene of IBDV JS strain combined with adjuvant Rg1-CpG couldsupply 91.7% protection rate after two immunization. Although we did not know the更多还原