为了探讨多杀性巴氏杆菌皮肤坏死毒素(PMT)对真核细胞的致病机制,将编码PMT的toxA基因以及其N端toxN(1～1461 bp)和C端toxC(2 958～3 858 bp)分别克隆到原核表达载体pET-32a中进行蛋白表达.3个重组蛋白在大肠杆菌中均以可溶性形式表达,其分子质量大小分别为176 ku、88 ku和67 ku,均能与His单克隆抗体发生特异性免疫反应.体外细胞毒性试验和豚鼠皮肤坏死试验均表明,rPMT与天然PMT具有相同的生物学功能.此外,以原核表达蛋白rPMT-N和rPMT-C分别制备的鼠多抗为一抗,对转染表达N端和C端真核质粒的Vero细胞进行IFA和Western-blot检测.结果表明,2种蛋白特异性良好,制备的多克隆抗体免疫反应条带单一.该研究的成功开展将为后续探究PMT细胞内作用机制提供可靠的生物材料.

Based on the proteomic analysis of avirulent and highly virulent strains of Haemophilus parasuis by Lable-free,four potential virulence proteins were screened and gene fragments were amplified.The target gene fragments were cloned into prokaryotic vector pET-32a to obtain the recombinant plasmids.The plasmids were respectively transformed into Rosetta strain to express recombinant proteins.After these proteins were purified by affinity chromatagraphy,mice were immunized with the recombinant proteins to produce polyclonal antibody.Western-blot results showed that the recombinant proteins have high immunogenicity and the prepared polyclonal antibodies are specific,which provides basis for investigation of the functions on the potential virulence factors from protein levels.

Porcine infectious anemia caused by Mycoplasma suis is a global disease and results in serious economic losses. To determine the prevalence of M. suis infection in eastern China, a cross-sectional serologic study was conducted with 3458 porcine serum samples randomly obtained from January 2014 to August 2016. The samples were tested with a blocking enzyme-linked immunosorbent assay developed in our laboratory. The seroprevalence was 33.3% in the complete sample set and was 25.9%, 37.8%, and 37.8% in 2014, 2015, and 2016, respectively. The seroprevalence was distinctly higher in summer (39.9%) and autumn (42.0%) than in spring (28.9%) and winter (23.3%). Shanghai was the region with the highest seroprevalence (54.2%) and Jiangsu the region with the lowest (23.0%). The seroprevalence was markedly higher in boars (47.1%), multiparous sows (47.0%), and replacement gilts (39.2%) than in piglets (24.2%), fattening pigs (17.2%), and nursery pigs (12.5%). These data demonstrate that the prevalence of M. suis infection is increasing yearly in eastern China.

Haemophilus parasuis (H. parasuis) is a common commensal Gram-negative extracellular bacterium in the upper respiratory tract of swine, which can cause Glässer's disease in stress conditions. Research on the pathogenicity of H. parasuis has mainly focused on immune evasion and bacterial virulence factors, while few studies have examined the interactions of H. parasuis and its host. Autophagy is associated with the replication and proliferation of many pathogenic bacteria, but whether it plays a role during infection by H. parasuis is unknown. In this study, an adenovirus construct expressing GFP, RFP, and LC3 was used to infect H. parasuis. Western blotting, laser confocal microscopy, and electron microscopy showed that Hps5 infection induced obvious autophagy in PK-15 cells. In cells infected with strains of H. parasuis differing in invasiveness, the levels of autophagy were positively correlated with the presence of alive bacteria in PK-15 cells. In addition, autophagy inhibited the invasion of Hps5 in PK-15 cells. Autophagy related genes Beclin, Atg5 and Atg7 were silenced with RNA interference, the results showed that autophagy induced by H. parasuis infection is a classical pathway. Our observations demonstrate that H. parasuis can induce autophagy and that the levels of autophagy are associated with the presence of alive bacteria in cells, which opened novel avenues to further our understanding of H. parasuis-host interplay and pathogenesis.

TGF-β1 plays an important role during the invasion of some bacteria into cells by regulating the expression of integrins, fibronectin, and other extracellular matrix proteins. We postulated that TGF-β1 levels could affect the invasion of Haemophilus parasuis into PK-15 cells. After infection by H. parasuis, PK-15 cells had elevated levels of TGF-β1 expression. Treatment of PK-15 cells with TGF-β1 prior to infection significantly decreased invasion by H. parasuis. Both TGF-β1 treatment and H. parasuis infection resulted in significant induction of fibronectin (Fn) and α5 integrin. Although pretreatment of PK-15 cells with siRNA fragments targeting Fn and α5 integrin resulted in enhanced H. parasuis invasion, H. parasuis attachment was elevated only on cells treated with the α5 integrin siRNA, there was no corresponding increase in attachment to cells treated with Fn siRNA. Our results firstly demonstrated that expression of TGF-β1, Fn and α5 integrin inhibited invasion of H. parasuis in PK-15 cells.

E. rhusiopathiae is the causative agent of erysipelas in animals and erysipeloid in humans, but its pathogenicity is poorly understood. To identify virulence factors associated with E. rhusiopathiae and screen engineered vaccine candidates, we used proteomics and transcriptomics to compare the highly virulent strain HX130709 with an isogenic avirulent derivative, HX130709a. 1,299 proteins and 1,673 transcribed genes were identified and 1,292 of the proteins could be associated with genes. In a comparison between HX130907 and HX130709a, 168 proteins and 475 genes exhibited differences in regulation level. Among these, levels for 61 proteins and transcripts were positively or negatively correlated. Gene Ontology (GO) analysis suggests that many of the down-regulated proteins in the attenuated strain have catalytic or binding functions. Potential protein-protein interactions suggest that some of the down-regulated proteins may regulate PTS, GMP synthase and ribosomal proteins. Morphological results showed that HX130709 and HX130709a have similar colony and capsule morphology. Growth curves and pyruvate measurements suggest that TCA cycle and saccharide phosphorylation levels were decreased and gluconeogenesis was increased in HX130709a. Our study confirms that SpaA and neuraminidase, but not hyaluronidase and capsule, are associated with virulence in E. rhusiopathiae. We conclude that the virulence of E. rhusiopathiae may be associated with slow reactions of the TCA cycle and down-regulation of selected proteins.

根据猪丹毒杆菌噬菌体基因测序结果,设计针对裂解酶(Ely)的特异性扩增引物,PCR扩增获得Ely产物,PCR产物经酶切后克隆人表达载体pET-28a(+),获得重组质粒pET-28a(+)-Ely,并转化至大肠杆菌BL21 (Rosetta)中.以1mmol/L IPTG在28℃诱导8h,SDS-PAGE结果表明,蛋白在上清中高效表达;通过Ni2+-NTA亲和层析法纯化目的蛋白,其纯度在90％以上;以100、10和1μg/mL的终浓度作用于猪丹毒杆菌培养物,每隔1h进行一次菌落计数,直至7h,结果表明该裂解酶在体外可以有效裂解猪丹毒杆菌,并呈一定的时间和剂量依赖性.该研究将为治疗耐药性猪丹毒杆菌感染提供新的思路.

为确定导致安徽和县某猪场猪只发病的病原,从该猪场送检病料(心、肺、脾、肝)中分离并纯化细菌,根据其形态特征、PCR鉴定及生化试验结果,确诊为猪丹毒杆菌.spaA基因序列分析结果显示该菌与猪丹毒杆菌G4T10(GenBank登录号:KF150604.1)有99％的同源性.全自动生化反应报告该菌为丹毒丝菌的概率高达95％.小鼠攻毒试验表明,试验组小鼠在腹股沟皮下注射该菌后,4d内全部死亡;死亡小鼠剖检可见全身败血性出血,各脏器均有病变,从死亡小鼠的血液、心、肝、脾、肺等组织中分离到与该感染菌株形态特征完全一致的细菌.药敏试验结果表明,该菌对β-内酰胺类和大环内酯类抗生素敏感,对链霉素、卡那霉素、四环素、多西环素、林可霉素和磺胺异恶唑耐受.