摘要
副猪嗜血杆菌(Haemophilus parasuis,HPS)是猪上呼吸道的常在菌。其致病菌在一定条件下可引起以多发性浆膜炎、关节炎和脑膜炎为主要特征的革拉泽氏病(Glasser’s Disease)。近年来,该病在保育猪群中的发生逐年上升,已成为危害养猪业的重要传染病之一,受到人们的日益重视。虽然近几年加大了该病原的研究力度,筛选出大量的毒力相关基因,但目前为止尚不十分清楚HPS的致病机理。
本研究利用重叠PCR(overlapping PCR)的方法构建了HPS hhdA基因的外源打靶基因Hup-Kan-Hdown。将其连接至自杀性质粒载体pDS132,获得了可在宿主菌E.coli SM10λpir中稳定遗传的外源打靶载体pDS-HPhhdA-Kan。将该打靶载体用电转法导入HPS中,经卡那霉素抗性筛选和蔗糖负筛选,得到HPS hhdA基因缺失株(HPS hhdA)。生化试验表明,该突变株的生化特性及NAD生长依赖性未见改变。生长曲线的测定表明该突变株的生长慢于标准株。豚鼠感染试验表明毒力变化不明显。说明hhdA基因不影响HPS的生长、繁殖,与其致病力无明显相关性。本试验建立的HPS遗传操作突变系统为实验室日后验证HPS各种基因功能奠定基础。
本研究对HPS的一种胞外丝氨酸蛋白酶(extracellular serine protease, esp)进行了序列分析并将该蛋白进行原核表达。HPS esp一级结构分析表明,前23个氨基可能为信号肽序列。C端转运单位氨基酸序列的系统进化树表明,该蛋白与流感嗜血杆菌中IgA蛋白酶的亲缘关系较近。承载结构域抗原表位的分析表明,50～57、95～108、120～130、185～196、230～241和421～425等6个氨基酸序列表位抗原的可能性较大。将克隆的esp基因连接至表达载体pET30a,构建了表达质粒pET-esp。将测序鉴定正确的重组表达质粒利用IPTG进行诱导表达。经大量诱导条件优化试验,得到了IPTG终浓度为0.6 mmol/L,32℃振荡培养6 h的最优表达条件。SDS-PAGE分析显示表达产物的分子量约为82 kDa。以该蛋白为抗原,加入等体积的206佐剂免疫豚鼠,3次免疫后以浓度为5×10~9 CFU/mL的菌液进行攻击试验,每只豚鼠注射1 mL。结果表明,esp可以部分保护HPS的感染(6/12),有望成为HPS病免疫诊断和疫苗研制的候选抗原。
Haemophilus parasuis (HPS) is a commensal organism of the upper respiratory tract of healthy pigs. Under appropriate conditions, this bacteria cause Gl?sser’s disease, characterized by fibrinous polyserositis, arthritis and meningitis. Gl?sser’s disease has recently emerged as one of the typical bacterial diseases in swine industry in the worldwide, which has increasingly attented by people. Although significant studies on HPS have been reported as well as several virulence factors have been screened, the pathogenesis is yet to understand.
In this study, the overlapping PCR method was used to construct the Hup-Kan-Hdown gene, an exogenous gene targeting gene of hhdA in HPS. When the recombinant gene was inserted into pDS132, the targeting plasmid, pDS-HPhhdA-Kan, was constructed, which was stable duplicated in E. coli harboringλpir gene. Electroporation of pDS-HPhhdA-Kan into HPS, through the screening with kanamycin resistance and 5% sucrose, HPS hhdA gene deletion strain (HPS△hhdA) was selected that could grow on plates with kanamycin resistance but not on plates containing 5% sucrose. Biochemical tests showed that the biochemical characteristics of the mutant strain did not change as well as the NAD-dependent growth. The growth curve showed that mutant strains growed little slower than standard strains. The virulence of the mutant strains did also not change significantly preformed by the guinea pig infection experiment. All those results showed that hhdA gene does not affect the growth of HPS and its reproduction. There was no significant correlation between virulence and hhdA gene. This established HPS mutation system opens the possibility for the functional analysis of genes involved in the infectious process of this animal pathogen.
The HPS esp was then sequenced, analysis and expression. The sequence analysis showed that the first 23 aa was signal peptide. The longest conserved sequence, E(M/V)NNLNKRMG(E/D)LR(G/D), was compared with the C-terminal of the serine protease family. The validation of 3-D structure of the C terminal translocator domain was showed the monomericβ-barrel containing 12β-strands and anα-helix in the N-terminal. Many distinct antigenic epitopes in the passger domain of HPS esp were identified by computation : 50～57, 95～108, 120～130, 185～196, 230～241 and 421～425. When the esp gene was inserted into pET30a, the pET-esp was constructed. After induced with Isopropylβ-D-1-Thiogalactopyranoside (IPTG) and optimized the conditions of expression, the esp fusion protein was highly expressed at 0.6 mmol/L IPTG, 32℃, 6 h. SDS-PAGE showed the molecular weight of about 82 kDa. To evaluate the immunogenicity, were immunized with esp vaccine contained equal volume 206 adjuvant. Then guinea pigs were challenged with 5×10~9 CFU per guinea pigs of HPS and partial protection. This result indicates that esp is good candidate immunogens that could be used to improve HPS vaccines.
引文
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