【作者】 李军星；

【导师】 姜平；

【作者基本信息】 南京农业大学 ， 动物检疫与动物源食品安全， 2009， 博士

【摘要】 副猪嗜血杆菌(Haemophilus parasuis, H.parasuis)是猪Glasser’s病的病原体,猪上呼吸道的一种共栖菌,它可在特定条件下侵入机体而引起以纤维素性多发性浆膜炎、关节炎和脑膜炎为特征的全身性疾病。目前,我国很多省市患病猪群中均可分离获得副猪嗜血杆菌,该菌已成为保育猪死亡的主要原因之一,给我国养猪业带来了巨大的经济损失。但由于不同血清型及同一血清型的不同菌株之间抗原性差异较大,交叉保护性差；同时该菌的毒力因子和保护性抗原尚不清楚,因此,目前尚无有效的预防和控制措施。猪繁殖与呼吸综合征是由猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus, PRRSV)引起的猪的一种高度传染病,又称“猪蓝耳病”,本病以妊娠母猪的繁殖障碍(后期流产、死胎、木乃伊胎、弱胎)及各种年龄猪特别是仔猪的呼吸道疾病(间质性肺炎)为特征。本病给世界养猪业造成巨大的经济损失,现已成为危害规模化养猪生产的主要疫病之一。目前,已有商品化的弱毒疫苗和灭活苗用于PRRS的防制,但由于其自身的缺陷而不能提供有效的免疫保护.近年来,我国大范围出现高致病性PRRSV感染,给我国养猪业造成重大经济损失,同时也对该病的防治带来新的挑战。本研究对我国6个省市的副猪嗜血杆菌的分子流行病学进行了调查,对其热休克蛋白70 (Heat shock protein 70, HSP70)进行了克隆鉴定,并设计构建了共表达副猪嗜血杆菌HSP70或其羧基端与PRRSV膜相关蛋白GP3和GP5的重组腺病毒,通过小鼠免疫试验证明了副猪嗜血杆菌HSP70及其羧基端的免疫增强作用,并通过攻毒保护试验证实了对高致病性毒株攻毒有一定的免疫保护效果,为PRRSV基因工程疫苗研究提供了新的思路。本研究内容分为以下6个部分：1.副猪嗜血杆菌tbpA基因PCR-RFLP分析利用针对H. parasuis转铁蛋白基因tbpA的PCR-RFLP分型方法,对2003-2008年分离自江苏、上海、广西、浙江、江西和安徽等6省市的57个H. parasuis分离株及15个参考菌株进行了PCR-RFLP分析,15个血清型参考菌株分为9种基因型,57个H. parasuis流行分离株分为15种基因型,其中在我国最为流行的基因型分别为DBN(38%),ABN(18%)与DBP(12%)。该结果证明副猪嗜血杆菌在我国猪群中普遍存在,并至少有15个RFLP基因亚型,从而为我国副猪嗜血杆菌病的防治提供了重要理论依据。2.副猪嗜血杆菌热休克蛋白70基因的测序、表达与抗原性鉴定利用简并引物首次从副猪嗜血杆菌基因组DNA中克隆获得全长HSP70基因(GenBank登录号为EU693116),DNA测序结果表明,HSP70完整阅读框1908bp,根据编码序列推导出相应635个氨基酸。经BLAST分析表明,其氨基酸序列与溶血性曼氏杆菌、杜克雷嗜血杆菌、胸膜肺炎放线杆菌等同源性最高,达到90%左右。将HSP70部分基因克隆到原核表达载体pET-32a(+)中,经酶切鉴定正确后转化感受态大肠杆菌BL21,以异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达融合蛋白。SDS-PAGE表明表达的融合蛋白约46KDa,用猪抗副猪嗜血杆菌血清进行Western-blotting,结果显示该融合蛋白具有副猪嗜血杆菌抗原性。用镍柱亲和层析法对重组蛋白进行纯化,纯化后的蛋白免疫小鼠,制备抗血清,Western-blot结果显示,小鼠抗血清与该融合蛋白有明显的特异性反应条带,证明该重组蛋白具有良好的抗原性。3.共表达H. parasuis HSP70与PRRSV GP3和GP5重组腺病毒的构建与鉴定利用PCR扩增出高致病性PRRSV-SY0608分离株的GP3基因,按正确的读码框与GP5基因串联,成功构建穿梭载体pShuttle-CMV-GP3-GP5,经PCR、测序鉴定正确。然后用两个不同linker将副猪嗜血杆菌的HSP70基因按正确的读码框克隆入pShuttle-CMV-GP3-GP5,构建另外两个穿梭载体pShuttle-CMV-HSP-GP3-GP5及pShuttle-CMV-HSP-2A-GP3-GP5.上述三个穿梭载体经PmeⅠ线性化后在BJ5183大肠杆菌内与腺病毒骨架载体pAdEasy-1同源重组,产生重组腺病毒基因组DNA。将重组腺病毒基因组DNA经PacⅠ酶切线性化后脂质体法转染HEK-293A细胞,分别在细胞内包装成完整的腺病毒rAd-GP35、rAd-HS35和rAd-HSA35。上述三个重组腺病毒的TCID50分别为10-10.0/mL、10-10.5/mL和10-10.25/mL。通过RT-PCR、间接免疫荧光试验(IFA)和Western blot可以检测到HSP/GP3/GP5蛋白的表达,证明重组腺病毒可以正确表达目的蛋白,从而为下一步的免疫试验打下基础。4.共表达H.parasuis HSP70与PRRSV GP3和GP5重组腺病毒的小鼠免疫特性研究取75只BALB/c小鼠随机分为5组,每组15只。第1、2组分别注射PBS或wtAd(野生型腺病毒)作为对照组,第3-5组分别背部皮下免疫重组腺病毒rAd-GP35(融合表达GP3-GP5蛋白)、rAd-HS35(融合表达HSP-GP3-GP5蛋白)和rAd-HSA35(共表达HSP-2A-GP3-GP5融合蛋白),间隔三周用同样方法加强免疫一次。分别在首次免疫后21d、35d及49d采血测定ELISA抗体及中和抗体,分离小鼠脾淋巴细胞经PRRSV抗原刺激后测定其增殖能力及分泌细胞因子的能力。结果为：3-5组小鼠在免疫后均可诱导PRRSV特异的体液和细胞免疫应答。与免疫rAd-GP35的小鼠相比,免疫rAd-HS35和rAd-HSA35的小鼠产生了更高滴度的中和抗体,二免后6周效价为1：64,并可产生较强的淋巴细胞增殖。同时,免疫rAd-HS35与rAd-HSA35的小鼠的脾细胞经体外PRRSV抗原刺激后产生了比rAd-GP35更高水平的IFN-γ(P<0.05)和IL-4。上述实验表明,副猪嗜血杆菌HSP70对所携带的抗原具有免疫增强作用。5.共表达H. parasuis HSP70与PRRSV GP3和GP5重组腺病毒对仔猪的攻毒保护研究取20头2-3周龄的PRRSV阴性猪,随机分为4组,每组5头,其中三组分别间隔三周免疫两次重组腺病毒rAd-GP35、rAd-HS35及rAd-HSA35,另外一组按同样方法免疫野生型腺病毒wtAd作为对照。在二免后三周用高致病性SY0608株PRRSV进行攻毒,以观察不同重组腺病毒对仔猪攻毒的免疫保护效力。分别在首次免疫后21d、42d、45d、49d和56d采血测定ELISA抗体、中和抗体、血清中细胞因子、病毒血症等。攻毒后每日观察并记录临床症状、测定体温,实验结束时对所有攻读猪进行剖检,观察病理变化。结果为：免疫rAd-GP35, rAd-HS35及rAd-HSA35的猪产生了PRRSV特异性的体液免疫应答,且rAd-HSA35诱导的中和抗体滴度明显高于rAd-GP35免疫组,差异显著(P<0.05).同时,rAd-HS35及rAd-HSA35免疫仔猪血清中的IFN-γ(P<0.05)及IL-4的含量也明显升高。攻毒后,与wtAd和rAd-GP35免疫组相比,rAd-HS35及rAd-HSA35免疫仔猪的临床症状较轻,病毒血症较低,肺部病理变化也较轻。此外,rAd-HSA35诱导的保护效力明显高于rAd-HS35.结果表明HSP70与GP3-GP5共表达可以增强PRRSV抗原的诱导的免疫应答并对同源毒株攻毒提供一定的免疫保护。其中,重组腺病毒rAd-HSA35免疫保护作用较好,从而为高致病性PRRSV基因工程疫苗研究奠定了基础.6.共表达H.parasuis HSP70羧基端与PRRSV GP3和GP5重组腺病毒的构建与小鼠免疫特性研究本研究利用PCR扩增出副猪嗜血杆菌的HSP70羧基端(384-635aa)基因tHSP70,然后用两个不同linker将副猪嗜血杆菌的tHSP70基因按正确的读码框克隆入重组穿梭载体pShuttle-CMV-GP3-GP5,构建另两个穿梭载体pShuttle-CMV-tHSP-GP3-GP5及pShuttle-CMV-tHSP-2A-GP3-GP5,按上述方法,构建了两个含tHSP70的重组腺病毒rAd-tHS35及rAd-tHSA35。RT-PCR、IFA和Western blot结果证明,该重组腺病毒可以正确表达tHSP/GP3/GP5蛋白,两个重组腺病毒病毒滴度均为10-12.0/mL。为进一步研究其免疫特性,取105只BALB/c小鼠,随机分为7组,每组15只。第1、2组分别注射PBS或wtAd作为对照组,第3-7组分别背部皮下免疫重组腺病毒rAd-GP35(融合表达GP3-GP5蛋白)、rAd-HS35(融合表达HSP-GP3-GP5蛋白)、rAd-HSA35(共表达HSP-2A-GP3-GP5融合蛋白)、rAd-tHS35(融合表达tHSP-GP3-GP5蛋白)和rAd-tHSA35(共表达tHSP-2A-GP3-GP5融合蛋白),间隔三周用同样方法加强免疫一次。结果为：第3-7组免疫后均可产生PRRSV特异的体液和细胞免疫应答。rAd-HS35、rAd-HSA35、rAd-tHS35和rAd-tHSA35免疫组中和抗体水平、淋巴细胞增殖反应、以及IFN-γ水平均显著高于rAd-GP35免疫组(P<0.05),但rAd-HS35、rAd-HSA35、rAd-tHS35、rAd-tHSA35四组之间并无显著差异(P>0.05)。该结果表明,副猪嗜血杆菌HSP70羧基端对所携带的抗原具有免疫增强作用,其佐剂作用与完整的HSP70相似。综上所述,副猪嗜血杆菌在我国发病猪群中广泛存在,基因型众多；其热休克蛋白70具有良好的抗原性；副猪嗜血杆菌HSP70或其羧基端(384-635aa)与PRRSV GP3、GP5蛋白共表达后,能明显提高其诱导的中和抗体水平和细胞免疫应答；共表达HSP70/GP3/GP5的腺病毒对同源毒株攻毒具有一定的免疫保护效果,为研究安全有效的PRRSV新型疫苗提供了新的思路和方法。更多还原

【Abstract】 Haemophilus parasuis (H.parasuis), the causative agent of Glasser’s disease, is a member of the family Pasteurellaceae and commensal organism of the upper respiratory tract of conventional pigs.Under appropriate conditions it can invade and cause severe systemic disease, characterized by fibrinous polyserositis, arthritis and meningitis. Currently, H.parasuis infection, which causing significant mortality and morbidity in piglets and great economic losses in pig industry, has been one of the most important swine diseases in China. However, Studies based on serotyping demonstrated that a high antigenic heterogeneity exists among H. parasuis strains; Moreover, virulence factors and protective antigens of H.parasuis are not known, so there is no universal vaccine to prevent and control the outbreaks of this disease.Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically significant viral diseases in the swine industry. It is characterized by reproductive problems in sows such as poor farrowing rates, premature farrowings, and increased stillbirths, as well as respiratory problems in piglets such as pneumonia and atrophic rhinitis. Current commercial PRRS vaccines, including both live attenuated and killed vaccines, have been wildly used, but they cannot provide effective protection against PRRS. Recently, the emergence and prevalence of highly pathogenic PRRSV strains across China, which led to great economic losses, bring new challenge to the prevention and control of the disease.In this study, H.parasuis field strains isolated from several provinces or municipality of China were genotyped and heat shock protein 70 (HSP70) gene was cloned from one of the strains; The recombinant adenoviruses expressing HSP70 or truncated HSP70 (tHSP70) and structural protein of PRRSV GP3/GP5 were constructed and the immonoenhancing property of HSP70 or tHSP70 was studied in mouse model by inoculation of mice with these recombinant adenoviruses; Protective efficacy of these recombinant adenoviruses against highly pathogenic PRRSV infection was examined in pigs, which provides new strategy for construction of PRRSV genetic engineering vaccine. The contents of the paper contain five parts as following:1. Characterization of Haemophilus parasuis field isolates from China by PCR-RFLP analysis of the tbpA geneFifty-seven of Heamophilus parasuis isolates along with 15 reference strains of all known serovars were subjected to PCR-FRLP (restriction fragment length polymorphism) based on tbpA gene. The analysis of the 1.9-kb tbpA amplicon using Taq I, Ava I and Afa I endonucleases produced 9 RFLP patterns for the 15 reference strains and 15 patterns for the 57 field isolates. And the first 3 prevalent genotypes in China were DBN (38%), ABN (18%) and DBP (12%). It is confirmed that Haemophilus parasuis existed widely in China with numerous genotype. It provides significant basis for prevention and control of the disease in China.2. Sequencing and expression of the HSP70 gene of Haemophilus parasuis and antigenicity of heat shock protein 70The HSP70 (dnaK) gene of Haemophilus parasuis was cloned and sequenced by PCR with designed primers, which having GenBank accession NO. EU693116。Nucleotide sequencing showed that the gene consists of an open reading frame of 1,908 bp and encodes a protein of 635 amino acids with a high degree of homology to that of Mannheimia haemolytica, Haemophilus ducreyi and Actinobacillus pleuropneumoniae, which were calculated to be 91%,91% and 87%, respectively. After cloning of the C terminal fragment of the HSP70 coding region into pET-32a(+), it was expressed in E. coli and confirmed by SDS-PAGE and Western-blotting using experimentally infected pig serum and mice serum immunized with purified recombinant HSP70. It could be used to study on the foundation of this protein in future.3. Construction and Identification of recombinant adenovirus expressing HSP70 of Haemophilus parasuis fused with GP3 and GP5 of Porcine reproductive and respiratory syndrome virusGenes of GP3 and GP5 were amplified from a highly pathogenic PRRSV isolate SY0608, and cloned into p-shuttle-CMV vetor tandem inframe which resulted in the recombinant pshuttle-CMV-GP3-GP5. Then, HSP70 gene of Haemophilus parasuis was colned into pshuttle-CMV-GP3-GP5 using two different linkers (5×Glycine or 2A gene of FMDV virus) beteen HSP70 and GP3gene, which leads to the production of recombinant pshuttle-CMV-HSP-GP3-GP5 and pshuttle-CMV-HSP-2A-GP3-GP5. Positive clones were identified by restriction enzyme ananlysis and further confirmed by sequencing. Recombinant plasmids were linearized with Pme I and homologously recombined with pAdEasy-1 backbone vector in BJ5183 host bacteria. The recombinant adenovirus DNA produced by recombination were linearized with Pac I and transfected into HEK-293 A cells for packaging of intact adenovirus. The expression of HSP70/GP3/GP5 by recombinant adenovirus in 293A cells were confirmed by RT-PCR, indirect immuno-fluorescence assay (IFA), and western blot. It shows that HSP70/GP3/GP5 could be expressed by recombinant adenovirus constructed in this study as expected, which provides the basis for the further immunogenicity experiment.4. Analysis of immunogenicity of recombinant adenovirus co-expressing HSP70 of H.parasuis and GP3-GP5 of PRRSV in miceIn this study, three replication-defective adenovirus recombinants were developed as potential vaccine against PRRSV in a mouse model. Three groups of BALB/c mice were inoculated subcutaneously twice at 3-week intervals with the recombinants expressing GP3-GP5 (rAd-GP35), HSP-GP3-GP5 (rAd-HS35) and HSP-2A-GP3-GP5 fusion protein (rAd-HSA35). Two additional groups were injected with wild type adenovirus (wtAd) or PBS as control. The results showed that the mice inoculated with recombinant adenoviruses developed PRRSV-specific antibodies, cellular immune response at 2 weeks post second inoculation. However, only mice immunized with recombinant adenovirus rAd-HSA35 developed significantly higher titers of neutralizing antibodies to PRRSV and produced stronger lymphocyte proliferation responses compared to mice immunized with rAd-GP35. It was also found that lymphocytes of mice immunized with rAd-HS35 or rAd-HSA35 were primed for significant higher levels of PRRSV specific IFN-y responses upon stimulation with purified PRRSV antigen in vitro than that of mice immunized with rAd-GP35. It suggested that HSP70 of haemophilus parasuis has the immuno-enhancing effect on the PRRSV antigens chaperoned.5. Analysis of protective efficacy of recombinant adenovirus co-expressing HSP70 of H.parasuis and GP3-GP5 of PRRSV against highly pathogenic PRRSV challenge in pigletsIn this study, twenty piglets were divided into 4 groups of five, and immunized twice with wtAd, rAd-GP35, rAd-HS35 and rAd-HSA35 with three weeks intervals, then challenged with SY0608 strain of highly pathogenic PRRSV by 3 weeks post boost. Immune responses of pigs immunized with these recombinant adenoviruses were detected and protective efficacy against homologous challenge were examined. The results showed that all animals vaccinated with rAd-GP35, rAd-HS35 and rAd-HSA35 developed specific anti-PRRSV ELISA antibody, neutralizing antibody. The humoral immune responses of rAd-HS35, especially rAd-HSA35 containing 2A of FMDV between HSP70 and GP3 gene, were significantly higher than that from rAd-GP35. Both IFN-y and IL-4 in serum of pigs were markedly induced by fusing with HSP70. Following challenge with PRRSV, piglets inoculated with recombinant rAd-HS35 and rAd-HSA35 showed lighter clinical signs, lower viremia and less gross lesion of lungs, comparing with those in rAd-GP35 group. Moreover, the protective efficiency induced by rAd-HSA35 was higher than that rAd-HS35. It indicated that HSP70 fused with GP3 and GP5 of PRRSV could markedly increase the immune responses and provide protection against virulent PRRSV challenge in pigs. The recombinant adenovirus rAd-HSA35 might be an attractive candidate vaccine for preventing and control of highly pathogenic PRRSV infections.6. Construction and immunogenicity of recombinant adenovirus expressing truncated HSP70 of Haemophilus parasuis fused with GP3 and GP5 of PRRSV in miceGene of tHSP70 (384-635 amino acid)was amplified from a H.parasuis isolate, and cloned into pshuttle-CMV-GP3-GP5 using two different linkers (5×Glycine or 2A gene of FMDV virus) beteen HSP70 and GP3gene, which leads to the production of recombinant pshuttle-CMV-tHSP-GP3-GP5 and pshuttle-CMV-tHSP-2A-GP3-GP5. Positive clones were identified by restriction enzyme ananlysis and further confirmed by sequencing. Recombinant plasmids were linearized with Pme I and homologously recombined with pAdEasy-1 backbone vector in BJ5183 host bacteria. The recombinant adenovirus DNA produced by recombination were linearized with Pac I and transfected into HEK-293 A cells for packaging of intact adenovirus. The expression of HSP70/GP3/GP5 by recombinant adenovirus in 293A cells were confirmed by RT-PCR, indirect immuno-fluorescence assay (IFA), and western blot. It shows that tHSP70/GP3/GP5 could be expressed by recombinant adenovirus constructed in this study as expected, which provides the basis for the further immunogenicity experiment. In order to detect the immunogenicity of the recombinant adenoviruses, five groups of BALB/c mice (15 mice per group) were inoculated subcutaneously twice at 3-week intervals with the recombinants expressing GP3-GP5 (rAd-GP35), HSP-GP3-GP5 (rAd-HS35), HSP-2A-GP3-GP5 (rAd-HSA35), tHSP-GP3-GP5 (rAd-tHS35), tHSP-2A-GP3-GP5 (rAd-tHSA35) fusion protein. Two additional groups were injected with wild type adenovirus (wtAd) or PBS as control. The results showed that the mice inoculated with recombinant adenoviruses developed PRRSV-specific antibodies, cellular immune response at 2 weeks post second inoculation. However, mice immunized with recombinant adenovirus rAd-HS35, rAd-HSA35, rAd-tHS35 and rAd-tHSA35 developed higher titers of neutralizing antibodies to PRRSV and produced stronger lymphocyte proliferation responses compared to mice immunized with rAd-GP35. It was also found that lymphocytes of mice immunized with rAd-HS35, rAd-HSA35, rAd-tHS35 and rAd-tHSA35 were primed for significant higher levels of PRRSV specific IFN-γresponses upon stimulation with purified PRRSV antigen in vitro than that of mice immunized with rAd-GP35. It suggested that truncated HSP70 of haemophilus parasuis has the immuno-enhancing effect on the PRRSV antigens chaperoned.In summary, H.parasuis exist widely in diseased pig herds with numerous genotypes; HSP70 of H.parasuis shows strong antigenicity; Fusion proteins HSP-GP3-GP5 and HSP-2A-GP3-GP5 expressed by recombinant adenoviruses could significantly improve both PRRSV-specific neutralizing antibodies and cell-mediated immune responses. Moreover, it is indicated that HSP70 fused with GP3 and GP5 of PRRSV could markedly increase the immune responses and provide protection against virulent PRRSV challenge in pigs. This study provides the foundation for developing the genetic engineering vaccine against PRRSV.更多还原