【作者】 张松林；

【导师】 金梅林； 陈焕春；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2008， 博士

【摘要】 1伪狂犬病毒鄂A株gE^-/gI^-基因缺失突变株的研究伪狂犬病是威胁世界养猪业的主要疾病之一。虽然美国和欧洲一些国家已经根除了该病,但是在中国和世界上许多其他国家,伪狂犬病每年仍然给这些国家带来巨大的经济损失。根除伪狂犬病的过程中,gE基因缺失疫苗配合相应的鉴别诊断方法至关重要的。本研究报道构建一种gE基因缺失疫苗株,并且通过动物试验来检验该疫苗株的免疫保护效果,拟研究适合种猪使用且安全高效的灭活疫苗。主要研究内容及结果如下:1.1伪狂犬病病毒鄂A株gE^-/gI^-基因缺失突变株的构建伪狂犬病病毒（Pseudorabies virus,PRV）属疱疹病病毒科,α疱疹病毒亚科猪疱疹病毒Ⅰ型,具有强烈的神经嗜性和潜伏感染性。糖蛋白基因gE、gI是PRV的主要毒力基因,它们与PRV在神经元中的传递有关。本研究首先以限制性内切酶STUⅠ、BSTEⅡ双酶切质粒pIECMV,得到gE/gI缺失转移质粒pIESE。序列分析表明,该质粒缺失了从gI基因StuI位点到gE基因BstEII位点共计1247 bp的片断。用gE/gI缺失转移质粒pIESE与PRV Ea基因组共转染猪肾传代细胞IBRS-2,经过空斑筛选获得PRV Ea gE^-/gI^-突变株。经Southern杂交,蛋白质斑点杂交试验和间接免疫荧光分析证明重组病毒的gE和gI基因已经通过同源重组方式被读码框不完整的gE和gI基因替换掉。1.2 PRV gE^-/gI^-基因缺失突变株免疫效果的研究1.2.1 PRV gE^-/gI^-基因缺失突变株对Balb/c小鼠免疫效果的研究28只4周龄的雌性Balb/c小鼠,随机分成4组,每组7只由脚趾分别免疫剂量为10⁵TCID₅₀的重组PRV gI^-/gE^-灭活疫苗,PRV gG^-/LacZ⁺灭活疫苗,Bartha株来源的商品化的gE^-灭活疫苗和100μL的DMEM,并于第28 d进行第二次免疫。所有组的小鼠通过肌肉注射方式注入剂量为2×10⁶ TCID₅₀PRV Ea株强毒。小鼠在免疫后的0、21、28、35、42、49和56采取血清作为免疫分析使用。PRV ELISA和中和抗体检测表明,所有免疫组在免疫后的第14 d都可以检测到PRV抗体,并且于二免后显著增长,在第42 d达到最高。gE缺失疫苗免疫组在免疫的各阶段没有检测到PRV gE-ELISA抗体。在攻毒实验中,PRV gI^-/gE^-和PRV gG^-/LacZ⁺免疫组没有出现临床发病症状,全部存活。而免疫商品化gE缺失疫苗组在攻毒的后的第3d出现明显的临床症状并在第5d全部死亡。DMEM对照组在攻毒后的第1d就出现强烈的神经症状并于攻毒后的第2d全部死亡。结果表明,10⁶ TCID₅₀ PRV Ea gE^-/gI^-能保护小鼠抵抗2×10⁶ TCID₅₀PRV Ea株强毒的攻击。1.2.2 PRV gE^-/gI^-基因缺失突变株对新生仔猪免疫效果的研究20头14日龄的PRV抗体阴性的仔猪被随机分成4组,分别通过肌肉注射方式免疫,免疫剂量为10⁵TCID₅₀的PRV gI^-/gE^-灭活疫苗,RV gG^-/LacZ⁺灭活疫苗,Bartha株来源的商品化的灭活疫苗和2 mL的DMEM。28d后以同样的途径和剂量进行二次免疫。分别与免疫后的0、21、28、35和42 d收集血清进行抗体分析。所有组的仔猪通过肌肉注射5×10⁶TCID₅₀的PRV Ea株强毒。PRV ELISA和中和抗体检测表明,所有免疫组在免疫后的第14 d都可以检测到PRV抗体。gE缺失疫苗免疫组在免疫的各阶段没有被PRV gE-ELISA检测到。在攻毒实验中,PRV gI^-/gE^-和PRV gG^-/LacZ⁺免疫组没有出现临床发病症状,全部存活。在免疫商品化gE缺失疫苗组,有2只仔猪在攻毒的后的第5d出现明显的临床症状明显并在第10d死亡。DMEM对照组在攻毒后的第2d就出现强烈的神经症状并于攻毒后的第7d全部死亡。结果表明,10⁵TCID₅₀PRVEa gE^-/gI^-能提供完全保护小鼠抵抗5×10⁶ TCID₅₀PRV Ea株强毒的攻击。2禽流感假病毒入侵模型的建立禽流感病毒的血凝素蛋白是病毒的表面抗原,具有免疫原性,可诱导抗体及细胞免疫,是决定宿主范围的因素之一。本研究将禽流感病毒H5N1亚型的HA蛋白整合到HIV-1构件的核衣壳内,包装成假病毒颗粒（HIV/H5-HA）,建立了模拟禽流感病毒入侵宿主的假病毒模型并将包装的假病毒免疫Balb/c雌性小鼠,从与受体结合,感染宿主细胞以及刺激机体产生保护性抗体3个方面对展示在HIV核衣壳表面的HA蛋白的功能进行验证。2.1 HIV/H5-HA的包装和检测根据A/chicken/Hubei/327/2004株AIV HA基因的核苷酸序列采用RT-PCR克隆了完整的HA基因序列至pcDNA3.1中,得到转移载体pBHHA。通过与含有HIV-1gag/pol构件的载体pCMV△8.2以及含有β-半乳糖苷酶报告基因的pHR’-CMVLacZ共转染293T细胞包装成假病毒HIV/H5-HA。Western blot检测的HA假病毒颗粒表达结果显示出3条特异性目的带,其大小与HA蛋白未经切割的前体蛋白HA0以及切割后形成的亚单位HA1和HA2大小相符,说明HA在假病毒颗粒表面得到了表达,并且表达的HA蛋白得到了正确的切割。通过流式细胞仪检测、电镜观察及细胞融合分析,进一步确定了HA蛋白成功的整合到HIV核衣壳内。通过LacZ染色,感染293T细胞,证明假病毒对细胞具有感染性。此外,为检验HIV/H5-HA是否如同天然禽流感病毒那样有着广泛的细胞嗜性,用HIV/H5-HA感染了MDCK等6种细胞,实验结果表明,HIV/H5-HA可以感染多种哺乳动物细胞。2.2 HIV/H5-HA模拟流感病毒入侵宿主细胞机制通过鸡红细胞凝集试验证明表达HA蛋白的HIV粒子可以与细胞表面的受体结合并使鸡红细胞发生凝集反应,其凝血效价达到2⁵。本研究通过不同浓度的NH₄Cl溶液与感染假病毒的的Vero细胞作用来模拟HIV/H5-HA在不同pH值下入侵细胞机制。结果表明,高pH值对HA假病毒进入宿主细胞具有明显的抑制作用,浓度为40 mmol/L的NH₄Cl可以完全抑制HIV/H5-HA对细胞的感染性:而且HIV/H5-HA对细胞的感染与NH₄Cl浓度成线性负相关关系（y=-0.028x+1.473,R²=0.960）,以上结果充分证明HIV/H5-HA是以一种pH依赖的方式感染宿主细胞的。同时也证明,禽流感假病毒模型可以作为禽流感病毒与宿主之间入侵和免疫应答等研究的技术平台。2.3 HIV/H5-HA对Balb/c免疫保护效果的研究5组雌性Balb/c小鼠,分别免疫不同剂量的HA假病毒,同时免疫灭活禽流感病毒和PBS注射组作为阳性对照和阴性对照。利用血凝抑制试验评价体液免疫效果。免疫一周后,HI抗体在假病毒免疫组被检测到,但在灭活疫苗对照组和阴性对照组没有检测出HI抗体。在免疫后的第二周,各免疫组的HI效价都迅速增长并在攻毒后的第一周达到最高值。在攻毒后的临床症状方面,阴性对照组的老鼠出现明显的临床症状（精神沉郁,反应迟钝,麻痹等）,并且体重较攻毒前减少了26.22%,最后在攻毒的6d内全部死亡。各免疫组在攻毒的第一周出现轻微的消瘦症状,并且在攻毒的第一周内平均体重减少12.78%-21.07%,但是平均体重在攻毒的第二周恢复到攻毒前的水平。以上实验数据显示,HA假病毒对老鼠具有良好的免疫保护效果。更多还原

【Abstract】 1 Study on a gE^-/gI^-mutant stainPseudorabies（Aujeszky’s disease） is one of the main diseases threatening the pig industry.Eradicating PRV in the United States and Europe has shown great progress,but PR is still an endemic problem in China and many other countries.In the process of controlling PRV,the ability to detect gE antibodies in infected pigs is crucial.In this study, we report the generation of a recombinant PRV virus with gI and gE gene deletions and the testing of its immunogenicity and protective immunity in vaccinated Balb/c mice and piglets.The main research was described an follows.1.1 Construction of PRV Ea gE^-/gI^-mutant stainPseudorabies virus（PRV） belongs toαherpesvirus family with the characteristics of neurotropism and strong latency,gE and gI are main virulent genes of pseudorabies virus. They all play important roles in the growth of PRV in nerve tissues.Transfer plasmid pIESE was generated by digesting the plasmid pIECMV with StuⅠand BstEⅡ.Sequence analysis dedicated there was about 1247bp deletion between StuⅠsite of gI gene and BstEⅡsite of gE gene.Genomic DNA of PRV Ea strain was co-transfected into IBRS-2 cells with transfer plasmid pIESE.After obvious cytopathic effect（CPE） was observed on day 2 post-transfection,the infected cells containing recombinant pseudorabies PRV Ea gI^-/gE^-viruses were harvested,frozen and thawed three times and virus isolated by plaque formation on IBRS-2 cells.The recombinant virus was confirmed by Southern blotting and indirect immunofluorescence assay（IFA）,and results showed the ORF of gI and gE genes had already been deleted by the way of homologous recombination.1.2 Analysis of immunization and virus challenge1.2.1 Immunization and virus challenge in miceFour groups of seven 4-week-old female Balb/c mice were each inoculated at footpad with either 10⁵ TCID₅₀ of recombinant PRV gI^-/gE^-vaccine,or 105 TCID₅₀ of PRV gG^-/LacZ⁺（Ea strain） vaccine,or 10⁵ TCID₅₀ of a commercial PRV gE^-（Bartha strain） inactivated vaccine or 100μL of DMEM.Mice were boosted once 28 days later with the same dose of inoculum respectively.Sera were collected at 0,21,28,35,42,49 and 56 days post vaccination for immunoassays.Mice then were challenged intramuscularly with 2×10⁶ TCID₅₀ of PRV Ea.After vaccination of mice,PRV ELISA and neutralizing antibodies were detected as early as 14 days after a single immunization in the sera of mice immunized with all of 3 of inactive vaccines.Neutralizing antibody titers increased significantly after the boost,and came to their peak value at day 42,then decreased between 42-49 days.No anti-gE antibodies were detected in mice immunized with PRV gI^-/gE^-or with commercial PRV gE^-at any of time point tested.No Antibodies to PRV and PRV gE were detected in control mice that received DMEM.The data showed that 10⁶TCID₅₀ PRV Ea gE^-/gI^-was effectively at protection 2×10⁶ TCID₅₀PRV Ea challenge. 1.2.2 Immunization and virus challenge in pigsTwenty 14-day-old piglets tested negative for PRV antibodies were randomly divided into four groups of five.Each group of piglets was inoculated intramuscularly with either 10⁵TCID₅₀ of PRV gI^-/gE^-vaccine or 10⁵TCID₅₀ of PRV gG^-/LacZ⁺ vaccine,or 10⁵ TCID₅₀ of PRV gE^-（Bartha strain） inactivated vaccine or 2ml of DMEM,and boosted once by the same dose and the same route 28 days later.Sera were collected at 0,21,28, 35 and 42 days post vaccination for immunoassays.All the pigs then were challenged intramuscularly with 5×10⁶ TCID₅₀ of PRV Ea strain.PRV ELISA,PRV gE-ELISA and microneutralizing assay were performed to assess antibodies against PRV.Antibodies to PRV could be detected by PRV ELISA and microneutralizing assay 14 days after vaccination.Anti-gE antibodies were not detected in 96-well microtiter plates coated with PRV gE monoclonal antibody in the PRV gE^-groups.In the virus challenge study,both vaccinated groups and control group were challenged with 5×10⁶ TCID₅₀ of the virulent PRV Ea strain.No clinical sign of PRV infection was observed with pigs vaccinated with recombinant virus PRV gI^-/gE^-and PRV gG^-/LacZ⁺,and all pigs of this group survived.In the other group vaccinated with commercial PRV gE^-,two pigs displayed obvious clinical symptoms（fervescence,diarrhea,disgorging,incoordination etc） on day 5 following challenge,and died on day 10 after challenge.Pigs in the DMEM control group showed neurological symptoms of PRV infection on day 2 following challenge and all died on day 7 after challenge.The HA protein is involved with receptor binding and eliciting immune responses, and also represents the best understood model system for cell entry.In this study,we incorporated hemagglutinin（HA） protein of H5N1 into HIV particles,and generated H5 hemagglutinin pseudotyped virus（HIV/H5-HA）,and evaluated its protection by virulent wild-type avian influenza virus（AIV） challenged in mice.1.2.3 The packaging and detecting of HIV/H5-HAHA gene of avian Influenza Virus（AIV） H5N1 subtype was amplified by RT-PCR and cloned into pcDNA3.1^（+） vector,then were cotransfected with retroviral vectors HIV-1 gag/pol encoded,pCMV△S.2 andthe HIV packagable vector encoding aβ-galactosidase （β-gal） reporter gene,pHR’-CMVLacZ into 293T cells.The HA protein expression could be verified by Western blot.Three main bands were seen which is consistent with the expected molecular weight for the full-length HA0 and the cleaved subunits HA1 and HA2.Further,HA protein expression was comfirmed by FACS,EM and cell-cell fusions. Infection activity of HIV/H5-HA were confirmed by LacZ staining and infecting 293Tcells.To test whether the AIV/H5-HA was infectious and displayed the similar host range as AIV,we infected 293T,BHK,Vero,PK-15,IBRS-2 and MDCK cell strains with HIV/H5-HA.The results showed that HA pseudotyping particles have wide cell tropism.1.2.4 Reproducing entering host cell mechanism of AIVIn the experiments,Hemagglutination and treatment with NH₄Cl tests were taken to assess the pathway of HIV/H5-HA entering host cell.For hemagglutination test, HIV/H5-HA was detected to make chicken erythrocyte agglutinate,and HA titer≧1:32. We treated the Vero cells with various concentration of ammonium chloride（0-50mM） before infection.The results displayed that treatment with 40mM NH₄Cl could cause complete inhibition of infection by HIV/H5-HA.There existed linear correlation between OD₄₂₀ value and the concentration of NH₄Cl for both of HIV/H5-HA（y=-0.028x+1.473, R²=0.960）.It showed that HIV/H5-HA entering the host cells perform a pH-dependant manner.All of above results revealwe developed a pseudotyped virus system for studying AIV entering host cell mechanism and immunologic response between AIV and host cells.1.2.5 Immunization and virus challenge studies in miceFive groups of six 6-week-old female Balb/c mice were injected intramuscularly,the first 3 groups injected with various HIV/H5-HA,while IA group injected with inactivated AIV and control group with 100μl PBS.HI was performed to detect antibodies in sera. HI antibodies could be detect in HIV/H5-HA inoculated groups but not found in both inactivated AIV group and PBS inoculated group on 7-day post-vaccination,and compared with IA and control groups,HI titers induced by HIV/H5-HA increased 2-4 fold.The mean HI titers increased significantly after immunization for all vaccinated groups in the following week,and reached a maximum were on 7 days after challenge.In the challenge experiments,mice in the control group showed severe signs of illness （apathy,paralysis）,and lost approximately 26.22%of their body weights,and died within 6 days post-challenge.All of the mice in vaccinated groups appeared slight sick within 7 days post-challenge.Body weights of mice in vaccinated groups lost approximately12.78%-21.07%within 7 days post-challenge,but regain their body weights to the pre-challenge level in the following week.HIV/H5-HA in this report were effective at protect mice against a lethal challenge by 10⁷ EID₅₀ of HPAIV H5N1 virus.更多还原