【作者】 李鹏；

【导师】 姜平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2011， 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十分清楚,因此,目前尚无有效的预防和控制措施。本研究对副猪嗜血杆菌的部分生物学特性及分子流行病学进行了研究,建立了P2-PCR检测方法,对副猪嗜血杆菌外膜蛋白P2（Out membarine protein P2, OMP P2）进行了克隆表达及ELISA抗原性试验,并通过攻毒保护试验证实了对高致病性毒株攻毒有较好的免疫保护效果。本研究内容分为以下5个部分：1.副猪嗜血杆菌的分离鉴定与部分生物学特性比较将血清5型副猪嗜血杆菌参考株HS-DY05接种固体培养基TSA、PPLO琼脂、巧克力琼脂、胰（?）蛋白胨琼脂和液体培养基TSB、PPLO液体、胰（?）蛋白胨和马丁肉汤,通过观察菌落大小、平板计数试验,结果证明TSA和TSB是该菌的最适培养基。采集123份临床疑似副猪嗜血杆菌病病例,通过病理剖解、TSA和TSB分离培养、和生化鉴定,分离获得111株HPS分离株。HPS HB070214接种于TSB,37℃、180r/min培养10～12h,菌数可达到109cfu/mL。通过革兰氏染色镜检和电镜观察,HPS分离株具有形态多样性,并在荚膜和菌毛上有较大差异。对分离菌用22种常规的抗菌药物进行了纸片法药敏试验,结果表明,高敏比例在50%以上的药物依次有头孢哌酮、头孢西丁、头孢他啶、复方新诺明、多粘菌素、替米考星、头孢噻呋和头孢曲松。乙酰甲喹、强力霉素、氟苯尼考、利福平、恩诺沙星和磺胺六甲氧嘧啶等常用药物均已出现50.0%以上的耐药菌株。将15种血清型HPS接种仔猪,观察其致病作用,结果为：HPS血清6、7、8和11型HPS毒力很弱或没有致病性,血清4、5、12和13型HPS则具有很强毒力或致病性,血清1、2、9、10、11、14和15型HPS显示出中等的致病力,与国外报道的HPS各血清型的毒力特点基本一致。将15种血清型HPS菌液灭活后,免疫家兔制备血清,结果为HPS琼扩试验抗体效价在1：8以上,15种血清型菌株之间的相互交叉免疫保护反应,表明不同血清型HPS全菌高免血清存在广泛的交叉免疫反应,不同血清型HPS存在共同抗原。2. ERIC-PCR技术用于副猪嗜血杆菌分离株基因分析采用肠杆菌基因间重复一致序列PCR方法,在对15种副猪嗜血杆菌血清型参考株鉴定获得15种不同ERIC-PCR指纹的基础上,对分离自中国东南部发生Glasser’s病的不同猪场的111株副猪嗜血杆菌进行了指纹鉴定。结果显示：111株分离株显示出23种指纹图谱,前三种最流行的指纹图谱为ERIC-PCR ⅩⅩ（20/111）, ⅩⅩⅢ（9/111）和Ⅳ（8/111）。且在111株分离株中,来自不同地区的分离株分别表现出不同种类的指纹图谱。该试验表明ERIC-PCR方法可适用于对某一地区的副猪嗜血杆菌进行分子流行病学的研究和基因型的鉴定；试验结果还揭示了副猪嗜血杆菌在中国东南部地区已广泛存在并具有多样的基因型。15种血清型的HPS参考株和111株HPS分离株的ERIC-PCR指纹图谱均显示各菌株均有一大小在1100bp左右的基因。该结果表明,中国东南部地区已广泛存在并具有多样的基因型,ERIC-PCR方法可用于副猪嗜血杆菌分子流行病学研究,同时,为今后寻找HPS的共同抗原基因提供了理论依据。3.副猪嗜血杆菌OMP P2基因序列测定与基因分型根据HB070412ERIC-PCR指纹图谱中特异基因的测序结果,利用已分离的菌株HB070412,根据NCBI上GENBANK中的HPS序列（ZP₀2477753）设计了一对引物,用PCR方法扩增了副猪嗜血杆菌外膜蛋白中的穿孔素前体蛋白P2基因（OMP P2）,并克隆到载体pMD18-T（T-Vector）,序列测定结果表明HB070412OMP P2基因全长1077bp,序列同源性分析表明该基因相当保守,与所报道的HPS OMP P2基因之间的核苷酸序列同源性为98.1%,氨基酸序列同源性为95.5%。以此序列设计引物对15种血清型HPS参考株和分离株AH080412、AH080526、GX080413、JS070421、QD080725、ZJ080625和HB070214进行了OMP P2基因的扩增产物测序和序列分析,结果表明所测菌株的OMP P2可分为2个基因型：Genotype Ⅰ知Genotype Ⅱ。Genotype Ⅰ的OMP P2基因大小在1077bp到1095bp之间,Genotype Ⅱ的基因大小在1167bp到1203bp之间,Genotype Ⅰ知Genotype Ⅱ在氨基酸水平具有92.8%的同源性,在核苷酸水平的同源性则为96.6%；在蛋白水平Genotype Ⅰ比Genotype Ⅱ的OMP P2少27-42氨基酸,Genotype Ⅰ的菌株均为致病性菌株,而Genotype Ⅱ的菌株则不具致病性。4.副猪嗜血杆菌重组OMP P2蛋白的高效表达与抗原性研究用pGEX-6p-1构建了原核表达载体pGEX-P2,在大肠杆菌中进行了高效表达,表达蛋白约占菌体总蛋白的50%,主要为包涵体形式。SDS-PAGE电泳结果显示表达的融合蛋白约为65kDa,Western-blot结果表明该表达蛋白具有抗原性。根据GenBank中副猪嗜血杆菌（HPS）的P2蛋白基因序列,设计合成1对特异性引物,用P2-PCR方法从HB070214株中克隆了大小为1077bp的P2蛋白基因。将P2蛋白基因亚克隆到原核表达载体pET-32a中构建了重组表达质粒pET-P2,在IPTG的诱导下成功表达了相对分子质量约为60500的可溶性融合蛋白P2-His;经SDS-PAGE和凝胶薄层扫描分析,融合蛋白的表达量约占细菌总蛋白量的30%。将融合蛋白加入镍琼脂糖凝胶树脂层析柱,经300mmo1/L咪唑磷酸盐缓冲液洗脱,其纯度可达92.5%。用Western blotting分析纯化后的融合蛋白,显示良好的生物学活性。利用纯化融合蛋白作包被抗原及优化EL ISA反应条件,建立了可检测抗HPS P2蛋白抗体的间接ELISA方法（P2-ELISA）,并将所建立的P2-ELISA与国外进口的HPS检测试剂盒Synbiocits-ELISA对196份临床送检血清进行平行检测,阳性符合率为93.4%。结果表明,本试验建立的P2-ELISA与Synbiocits-ELISA具有同样高的特异性。小鼠免疫试验结果表明OMP P2蛋白对小鼠的攻毒保护率为80%。5.副猪嗜血杆菌OMP P2基因PCR检测方法的建立与应用通过反应条件的优化、特异性和敏感性的测定,建立了一种快速、简便、准确的PCR方法,用于检测副猪嗜血杆菌（HPS） OMP P2基因。PCR扩增片段大小为1080bp左右,最小检测量为1000个HPS。与胸膜肺炎放线杆菌、多杀性巴氏杆菌、大肠杆菌、肠沙门氏菌、金黄色葡萄球菌和链球菌无特异性反应。用该法检测人工感染猪的肺脏、心脏和肾脏抽提的DNA,结果为阳性；检测肝脏、脾脏、脑组织样品抽提的DNA,结果为阴性。用该法检测人工感染发病仔猪及自然感染仔猪肺组织,并结合细菌分离培养,证明以肺脏组织为病料,通过细菌分离培养,再用PCR法鉴定分离菌体,可有效地用于HPS的特异诊断。检测116份来自发病猪场的患病仔猪肺脏病料,其中有110份为阳性,与16S rRNA PCR检测结果一致,表明该方法可用于Glasser’s病病原学检测。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 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, became one of the most important swine diseases in China. And it has demonstrated that a high antigenic heterogeneity exists among H. parasuis strains. But the virulence factors and protective antigens of H.parasuis are not clear, and there is no universal vaccine to prevent and control the outbreaks of this disease.In this study,111H.parasuis field isolates from several provinces or municipality of China were genotyped by ERIC-PCR and the biological characters of H.parasuis isolates were detected. And the OMP（out membrane protein） P2gene of H.parasuis was cloned and expressed, and the antigenicity of the OMP P2protein was identified. The protective efficacy of the recombinant P2proteins against highly pathogenic H.parasuis strain HB070214infection was examined in Balb/c mice, which provided new strategies for the diagnosis and development of H.parasuis genetic engineering vaccine. The contents of the paper including five parts as following:1. Isolation, identification and biological characters of Haemophilus parasuisFor isolation of Haemophilus parasuis several media, including trypone soya agar （TSA）, chocolate agar, pleuropneumonia-like organism（PPLO） agar and proteose peptone tryptone agar or into liquid media including tryptone soya broth（TSB）, Brian heart infusion（BHI）, Martin’s broth, pleuropneumonia-like organism（PPLO） broth and peptone tryptone, and the optimal media TSA and TSB were selected and compared in this experiment. The results showed that TSA and TSB could be used to culture the H.parasuis isolates. And then111strains of Haemophilus parasuis were isolated from clinical ill piglets. Haemophilus parasuis HB070214was inoculated into TSB and cultured for10-12hours under the condition of37℃,180r/min, the bacterial number was up to109cfu/mL. The morphological diversity and diference of capsules and pili of HPS were observed under electron microscopy. Susceptibility of the isolates was defected by using22antimicrobial. And the results showed that the drugs, which were susceptible to more than50%isolates, were cefoperazone, cefoxitin, ceftazidime, cotrimoxazole, polymyxin, tilmicosin, ceftiofur and ceftriaxone. Acetyl flumequine, doxycycline, florfenicol, rifampicin, enrofloxacin and sulfa six SDM appeared no susceptibility to more than50%isolates. Pig experiment results showed that the HPS serumtype6,7,8and11had very low or no virulence, while the HPS serumtype4,5,12and13has very strong virulence. And HPS serumtype1,2,9,10,11,14and15showed moderate pathogenicity. The standard Haemophilus parasuis which were maintained at Nanjing Agricultural University were cultured and inactived antigens were prepared, and the antibodies to HPS were made by the antibody titers were more than1:8Gel Diffusion （GD） test. And the cross-immune responses existed between the15serotypes of HPS and their hyperimmunized serum.2. Genotype analyzing of Haemophilus parasuis field isolates with ERIC-PCRThe fifteen serovars of H. parasuis reference strains were analyzed by Enterobacterial Repetitive Intergenic Consensus PCR （ERIC-PCR）. The results showed that all the15serovars of reference strains gave unique, reproducible ERIC-PCR fingerprints. The results of the111field isolates from South-east of China showed that they presented23special fingerprints. The first3prevalence of ERIC-PCR fingerprint type were ⅩⅩ（20/111）, ⅩⅩⅢ（9/111） and Ⅳ（8/111）. And all provinces had different fingerprint types of H. parasuis. A gene about1100bp in the ERIC-PCR fingerprints was found in all15serotypes HPS reference strains and111HPS isolates. It indicated that H. parasuis has existed widely in South-east of China with large genotypes, and ERIC-PCR could be used to evaluate the molecular Epidemiology of H. parasuis and it provides theory basis to find HPS common antigen gene.3. Sequencing and genoping of Haemophilus Parasuis based on OMP P2GeneBased on the results of the ERIC-PCR, the fragment of about1100bp of strain HB070214of HPS was cloned into pMD18-T. The sequencing results indicated that this gene had1077bp length and the sequence was snbmitted to Genbank（GU323686）. The result of sequence analysis showed that it has95.5-98.1%homology with OMP P2gene. A pair of specific primers were designed based on the sequences of the OMP P2gene of Haemophilus Parasuis in Genbank, and the OMP P2genewere amplified from22isolates. And the sequences results showed that the OMP P2genes of the isolates could be divided into2genotypes: genotype Ⅰ and genotype Ⅱ. The sizes of genotype Ⅰ OMP P2gene was1077bp to1095bp, and genotype Ⅱ gene was1167bp to1203bp. The genotype Ⅰ and genotype Ⅱ have92.8%homology at the amino acid level and96.6%homology at the nucleotide level; Genotype Ⅰ had27-42amino acid deletion compared with Genotype Ⅱ at the protein level. The pathogenic strains serotype4,5,12,13belong to genotype I strains and the no pathogenic strain serotypes6,7,8and11belong to genotype Ⅱ. IIt indicated that this genotyping might be related to the virulence of the isolates.4. Expression and antigenicity of OMP P2fusion protein in E.coli.The OMP P2gene was ligated into pGEX-6p-1to get the expressing vector of pGEX-P2which was then transformed into E. col i BL21. The result of SDS-PAGE shows the expressed protein is about65kD, and the recombinant protein occupid persent50%of total cell protein and in inclusion body form. Western-blot results indicate that this protein possesses biological activity.A pair of specific primers was designed and synthesized according to the OMP P2gene of HPS in GenBank （ZP₀2477753）, And the OMP P2P2gene（1077bp） was amplified by P2-PCR from HPS strain HB070214, and cloned into pET-32a vector. pET-P2was identified by PCR and sequencing. Then the plasmid pET-P2was transformed into E.coli BL21（DE3）. The P2-His fusion protein with molecular weight of,60500Da was expressed by the induction of1.0mmol/L IPTG. It could specifically react to the antiserum against HPS by Western blotting. The P2-His fusion protein was further purified by nickel-agarose gel resin chromatographic column with300mmol/L imidazole phosphate elution buffe. The purity of P2-His was up to92.5%. The P2-His fusion protein was used as a packaging antigen, and a novel HPS P2-ELISA was established. The comparison results between P2-ELISA and kit Synbiocits-ELISA showed that they had93.4%agreement in the detecting results of196sera samples. It indicated that the indirect P2-ELISA had the same specific and sensitive as the Synbiocits-ELISA kit. In addition, The animal test results showed that this recombinant protein had80%protection rate in Balb/c mice.5. Application and Development of PCR Assay for detection of OMP P2Gene of Haemophilus parasuisA PCR assay for amplifying OMP P2gene of Haemophilus parasuis（HPS） was developed for quick and accurate detection of HPS. The results showed that the size of the amplified PCR product was approximately1080bp. The required minimum dose of HPS was1000bacteria. The specificity of the PCR was tested on Actinobacillus pleuropnemoniae, Pasteurella multocida, E coli, Salmonella enterica, Staphylococcus aureus and Streptococcus, the results showed that it did not recognize these bacteria. The positive results were obtained by amplifying the extracted DNA from the artificial challenged piglets. And the negative results were obtained from the natural infection. Twelve clinical samples （6from experimentally infected piglets with HPS isolates and6from naturally infected piglets） were tested by PCR. The results showed that the PCR assay could be used to detect HPS from lung sample. The lung samples of sick piglets from116pig farms were detected, the results showed that the110samples of them were positive to HPS, which were consist with the results as16S rRNA PCR test. It indicated that the P2-PCR assay could be used to detect HPS, specially in the Glasser’s disease.更多还原