从噬菌体随机十二肽库中筛选新城疫病毒血凝素-神经氨酸酶模拟表位

以生物素标记的抗新城疫病毒(NDV)血凝素-神经氨酸酶(HN)的单抗M22为分子探针,从噬菌体随机12肽库中筛选鉴定该抗体所识别的抗原模拟表位.经过三轮亲和筛选,得到了四个能与单抗M22反应的阳性噬菌体单克隆,分别为CLONE 11、17、18、20.竞争ELISA试验表明,CLONE 20与M22的结合能被新城疫弱毒株La Sota特异性抑制,抑制率达67.3%.经测序,获得CLONE 20的插入序列为SWFHHHQARAPM,同源性分析认为WF和QAR在HN的抗原性中起重要作用.动物试验结果表明,CLONE 20能诱导SPF鸡产生一定水平的具有血凝抑制活性的特异抗体.以上结果显示SWFHHHQARAPM是具有免疫原性的HN抗原模拟表位.     

2株B型副鸡禽杆菌的分离鉴定

2010年3月、2011年12月分别从北京平谷、延庆疑似鸡传染性鼻炎的病鸡体内各分离到1株细菌,根据发病鸡群的临床症状、细菌分离培养、形态观察、过氧化氢酶试验、革兰氏染色结合动物回归试验、免疫攻毒试验和PCR等方法初步鉴定为副鸡禽杆菌(Apg);同时将分离菌株送匈牙利诗华研发中心进行分型鉴定,确定为B型副鸡禽杆菌,根据分离地点将分离菌株命名为B型副鸡禽杆菌(平谷株和延庆株).通过GenBank与已经发表的Apg基因序列进行同源性比对分析,结果显示,平谷株、延庆株基因序列同源性达到99.6％,而与参考株Modesto的基因核苷酸序列同源性达到98.3％、98.8％.     

Identification of antigenic epitopes of the SapA protein of Campylobacter fetus using a phage display peptide library

In this study, we immunized mice with prokaryotically expressed recombinant surface layer protein, SapA, of Campylobacter fetus, generated hybridomas secreting mouse monoclonal antibodies (mAb) targeting SapA, and purified the mAb A2D5 from mouse ascites using saturated ammonium sulfate solution. The mAb A2D5, coated onto ELISA plates, was used to screen the phage random 12-peptide library through three rounds of panning. Following panning, 15 phage clones were randomly chosen and tested for reactivity with mAb A2D5 by indirect ELISA. Single-stranded DNA from positive clones was sequenced and compared with the sequence of SapA to predict the key epitope. ELISA and/or Western blot analyses further validated that synthetic peptides and recombinant peptide mimotopes all interact with mAb A2D5. Nine of ten positive phage clones identified by screening were sequenced successfully. Seven clones shared the same sequence HYDRHNYHWWHT; one had the sequence LSKNLPLTALGN; and the final one had the sequence SGMKEPELRSYS. These three sequences shared high homology with SapA J05577 in the region GNEKDFVTKIYSIALGNTSDVDGINYW, in which the underlined amino acids may serve as key residues in the epitope. ELISA and/or Western blot analyses showed that mAb A2D5 not only interacted with the four synthetic peptide mimotopes, but also with 14 prokaryotically expressed recombinant peptide mimotopes. The mimotopes identified in this study will aid future studies into the pathological processes and immune mechanisms of the SapA protein of C. fetus.     

Identification of antigenic epitopes of the SapA protein of Campylobacter fetus using a phage display peptide library

In this study, we immunized mice with prokaryotically expressed recombinant surface layer protein, SapA, of Campylobacter fetus, generated hybridomas secreting mouse monoclonal antibodies (mAb) targeting SapA, and purified the mAb A2D5 from mouse ascites using saturated ammonium sulfate solution. The mAb A2D5, coated onto ELISA plates, was used to screen the phage random 12-peptide library through three rounds of panning. Following panning, 15 phage clones were randomly chosen and tested for reactivity with mAb A2D5 by indirect ELISA. Single-stranded DNA from positive clones was sequenced and compared with the sequence of SapA to predict the key epitope. ELISA and/or Western blot analyses further validated that synthetic peptides and recombinant peptide mimotopes all interact with mAb A2D5. Nine of ten positive phage clones identified by screening were sequenced successfully. Seven clones shared the same sequence HYDRHNYHWWHT; one had the sequence LSKNLPLTALGN; and the final one had the sequence SGMKEPELRSYS. These three sequences shared high homology with SapA J05577 in the region GNEKDFVTKIYSIALGNTSDVDGINYW, in which the underlined amino acids may serve as key residues in the epitope. ELISA and/or Western blot analyses showed that mAb A2D5 not only interacted with the four synthetic peptide mimotopes, but also with 14 prokaryotically expressed recombinant peptide mimotopes. The mimotopes identified in this study will aid future studies into the pathological processes and immune mechanisms of the SapA protein of C. fetus.     

Identification of Avibacterium paragallinarum serovar B-1 from severe infectious coryza outbreaks in Panama

The isolation and identification of Avibacterium paragallinarum serovar B-1 from severe infectious coryza outbreaks in broiler breeders in Panama is reported for the first time. Infectious coryza was absent for over a decade in the breeder farms area. Disease outbreaks were characterized by an up to 45% drop in egg production and increased mortality. Use of a commercial trivalent bacterin and a strengthened biosecurity program prevented outbreaks in susceptible flocks in the farm.     

Immunogenicity and haemagglutination of recombinant Avibacterium paragallinarum HagA

Inactivated vaccines of Avibacterium paragallinarum provide protection and reduce the economic losses caused by infectious coryza. However, inactivated bacterins provide protection only against the Page serovars included in the vaccine. In this study, we investigated the immunological properties of a functional recombinant haemagglutinin protein (rHagA) derived from a Taiwan isolate strain A9 as the immunogen for vaccination. The rHagA subunit vaccine protected 71% of immunized chickens against 10(10) colony-forming units (CFU) of viable A9. Vaccinated chickens which showed no clinical signs of coryza developed haemagglutination inhibition (HI) titers of 1:10 or greater. Haemagglutination (HA) of serovars A and C was not affected by the presence of rHagA specific antiserum. The HA of rHagA could only be induced against formaldehyde-fixed chicken red blood cells (FA-RBCs). These results suggested that HagA is a moderate immunogen and might not be a major haemagglutinin in vivo. However, HagA might be involved in haemagglutination when treated serovar C aggregates fixed RBCs in vitro.     

副鸡禽杆菌荚膜多糖的提取及HPLC分析

为了研究副鸡禽杆菌的荚膜多糖的构成及生物学活性,本试验提取了B型副鸡禽杆菌的荚膜多糖,测定其含量,并分析单糖组分及分子量大小。用乙醇沉淀法在细菌培养液上清中提取了荚膜多糖,利用25%乙醇去核酸、苯酚抽提去蛋白,最后以苯酚-硫酸法测定其含量,高效液相色谱(HPLC)分析其组分及分子量大小。结果显示,每升菌液可以抽提得到荚膜多糖85mg,含量为59.6%,该多糖主要由L-鼠李糖、D-木糖、D-葡萄糖等单糖组成,总分子量约209Ku。     

Identification and expression of a gene encoding an epitope that induces hemagglutination inhibition antibody to Avibacterium paragallinarum serovar A

The aims of this study were the identification, cloning, and expression of a genetic region encoding an epitope that induces hemagglutination inhibition (HI) antibody against Avibacterium paragallinarum serovar A and an evaluation of the recombinant protein for immunogenicity in chickens. Although two monoclonal antibodies (MAbs) with HI activity, designated S24-951 and S7-1716-5C, were generated in this study, no reactive proteins with both MAbs were identified by Western blot analysis. A gene fragment of 5157 bp, designated hpa5. 1, was cloned from genomic DNA, and a recombinant protein expressed by hpa5.1, designated HPA5.1, reacted with both MAbs on dot-blot analysis. HPA5.1 showed no hemagglutinating activity, but significantly absorbed HI antibodies in the chicken immune serum. Analysis using a series of deletion mutants prepared from hpa5.1 indicated that a 4.8 kbp gene in hpa5.1 is essential for the expression epitope recognized by MAb S24-951. In addition, chickens immunized once with HPA5.1 showed a high protection rate with sufficient HI antibody titers against challenge exposure with a virulent strain of A. paragallinarum serovar A strain 221. These results show that hpa5. I1 is responsible for the expression of an epitope that induces HI antibody, and HPA5.1 might be a candidate for the development of a new vaccine against avian infectious coryza caused by A. paragallinarum serovar A.     

Evaluation of a nanobody phage display library constructed from a Brucella-immunised camel

Brucella are invasive gram-negative bacteria that multiply and survive within eukaryotic cells causing brucellosis. Syrian (and Middle East) health and economy sectors are still affected by this disease causing a serious national problem that needs to be solved. Here, a strategy was developed to introduce a new generation of binders, known as Nanobodies (Nbs) in our combat against Brucella. These Nbs, recombinant single-domain variable fragments derived from camelid heavy-chain antibodies are very stable and highly soluble, making them a useful tool in numerous biotechnological and medical applications. In this work and without having access to purified antigens (Ags), a camel was immunised successfully with heat-killed Brucella melitensis strain Riv1 as demonstrated by the high titer of Ag-specific heavy-chain antibodies in the serum. Lymphocytes of the immunised camel were isolated and their Nb genes were cloned in a relatively large library of 10(8) individual transformants, of which 81% contained an insert with the proper size of a Nb gene. Phage display expression of the Nbs from this library and pannings on the Brucella lysate resulted in a clear enrichment of three distinct Nb-displaying phages (phage-Nbs), referred to as NbBruc01, 02 and 03, with specificity for Brucella. Producing these binders in a pure, soluble form, as well as identifying their specific targets, which are likely to be immunodominant Ags in Brucella, is expected to open wide perspectives for following the vaccination, diagnosis and treatment of brucellosis.     

法氏囊活性五肽BP5特异性结合肽的筛选及鉴定

为鉴定与具有免疫调节功能和抗肿瘤潜能的法氏囊活性五肽(BP5)特异性结合的多肽,本研究利用噬菌体展示技术,以BP5-BSA为靶分子,对噬菌体随机12肽库进行4轮亲和筛选,结合ELISA鉴定和竞争抑制试验,筛选出3个能够特异性与BP5结合的噬菌体阳性克隆.测序结果显示,其编码的12肽序列分别为:PINMQTLNCMAA(P2-12)、GCTLNPMSDLLG(P6-12)和MSSTLNGMLNSL(P7-12),其核心基序为TLNXM.通过人工合成P2-12、P6-12、P7-12多肽,并采用MTT法检测其对BP5抗肿瘤细胞增殖能力的影响,结果显示,P2-12和P7-12肽在0.2 μg/mL～20 μg/mL浓度下,P6-12肽在2μg/mL～20 μg/mL浓度下均能够抑制BP5抗小鼠WEHI-231 B淋巴瘤细胞增殖作用,其中P2-12在20 μg/mL浓度下抑制作用最为显著(p＜0.01).此外,p53荧光素酶活性检测结果显示,这3种BP5结合肽在实验浓度下均能够下调BP5促p53基因转录的活性.以上结果表明,这3种BP5特异性结合肽具有下调BP5抗肿瘤活性.本研究为进一步研究BP5抗肿瘤细胞增殖作用的机制提供了相关的实验依据.     

安徽省白羽肉鸡中副鸡禽杆菌的分离鉴定及流行病学分析

本研究试图通过病原分离对安徽省某大型白羽肉鸡养殖公司持续出现疑似鸡传染性鼻炎的鸡群进行确诊,从不同批次发生鸡传染性鼻炎疑似症状的鸡体内分离细菌,经细菌分离、纯化、回鸡试验,共分离到8株细菌,经形态及培养特性、生化试验、血清学特性、16S rRNA基因序列分析等方法对分离的细菌进行鉴定。结果表明所分离的细菌有1株为A型副鸡禽杆菌,7株为B型副鸡禽杆菌,说明在安徽省某地持续引起幼龄肉鸡发生鸡传染性鼻炎的流行中B型致病株是优势毒株,显示新的流行特点。     

ERIC-PCR genotyping of emergent serovar C-1 isolates of Avibacterium paragallinarum from Mexico

Between 2008 and 2010, 14 isolates of Avibacterium paragallinarum were identified as serovar C-1 in Mexico. All isolates were obtained from commercial laying hens suffering infectious coryza despite a history of vaccination. The enterobacterial repetitive intergenic consensus-based PCR genotyping showed that all isolates had a common pattern. Until recently, serovars A-1, A-2, B-1, and C-2 were the serovars prevalent in Mexico. Serovar C-1 has been identified in Japan and recently in the Americas in Ecuador. Our current study suggests that Av. paragallinarum serovar C-1 is an emerging serovar in Mexico. Our results also indicate that the Mexican isolates of Av. paragallinarum serovar C-1 may have a clonal relationship. Knowledge of the genetic diversity of Av. paragallinarum may be of value in understanding vaccine performance and identifying the best combination to achieve broader protection.     

东方田鼠肝脏T7噬菌体展示cDNA文库的构建

本项研究的目的是构建东方田鼠肝脏T7噬菌体展示cDNA文库,为筛选东方田鼠抗血吸虫病抗性相关基因奠定基础.用TRIzol试剂提取东方田鼠肝脏总RNA,分离纯化mRNA,经反转录合成双链cDNA.在双链cDNA末端加上EcoRⅠ/HindⅢ定向接头并用EcoRⅠ和Hind Ⅲ酶切,使其两端分别带EcoRⅠ和HindⅢ粘性末端.用Mini Column纯化、收集300 bp以上的双链cDNA片段,再连接于带有EcoRⅠ和HindⅢ末端的T7 Select 10-3b载体,经体外包装后,以BLT5403为受体菌构建T7噬菌体展示cDNA文库.经测定,库容量为1.3×107 PFU/mL,扩增后文库滴度为1.8×1011 PFU/mL.对从原始文库中随机挑取的100个噬菌斑进行PCR鉴定,重组率为91.7%,阳性克隆片段大小分布在200 bp～1 000 bp,其中有95.5%的插入片段大于300 bp.用日本血吸虫童虫可溶性抗原对文库进行了初筛,得到了21个ESTs,将这些阳性噬菌体克隆和血吸虫童虫共培养,其中大部分克隆诱导的童虫死亡率比阴性噬菌体对照高出2%～13%.     

Development of a multiplex PCR and PCR-RFLP method for serotyping of Avibacterium paragallinarum

Avibacterium (Haemophilus) paragallinarum (A. paragallinarum) is a causative agent of infectious coryza in chickens and is classified into three serovars by agglutination tests. In an effort to identify the serovars easily, PCR and PCR-RFLP were employed. As the target gene for PCR, the hypervariable region of HMTp210, which encodes the HA antigen, was used. PCR using primer sets around the hypervariable region amplified 0.8, 1.1 and 1.6 kbp fragments for serovars A, B and C, respectively. Alternatively, the 1.6 kbp fragments were amplified with another primer pair encompassing the hypervariable region and was subjected to digestion with Bgl II, which resulted in the detection of serovar-specific digestion patterns. These results indicate that PCR and PCR-RFLP using the hypervariable region of HMTp210 are alternative methods to identify the serovar of A. paragallinarum.     

Antimicrobial susceptibility, plasmid profiles and haemocin activities of Avibacterium paragallinarum strains

In this study, 18 Avibacterium paragallinarum isolates collected in Taiwan from 1990 to 2003 were serotyped and tested for resistance to antimicrobial agents. Serotyping revealed that 13 isolates were Page serovar A and 5 isolates were Page serovar C. More than 75% of the isolates were resistant to neomycin, streptomycin and erythromycin. The most common resistance pattern (15 isolates, 83.3%) was neomycin-streptomycin. Furthermore, 88.9% of the isolates were resistant to two or more antibiotics. About 72% of isolates contained plasmids (pYMH5 and/or pA14). Plasmid pYMH5 encoded functional streptomycin, sulfonamide, kanamycin and neomycin resistance genes and revealed significant homology to a broad host-range plasmid, pLS88. Plasmid pA14 encoded a putative MglA protein and RNase II, both of which might be associated with virulence. Furthermore, seven isolates showed haemocin activity. Plasmid pYMH5 is the first multidrug-resistance plasmid reported in A. paragallinarum and it may facilitate the spread of antibiotic-resistance genes between bacteria. The putative virulence plasmid pA14 and haemocin-like activity in A. paragallinarum indicate two possible mechanisms which might be responsible for the pathogenesis.     

三肽囊素抗独特型ScFv噬菌体展示文库的构建

利用在体外构建的三肽囊素抗独特型抗体可变区VH和VL基因,通过重叠延伸反应（SOE）,以（Gly4Ser）3为连接肽,将VH和VL基因连接成为VH-Linker-VL ScFv,且ScFv DNA与噬菌粒载体pHENI的连接产物转化于大肠杆菌TGl,经辅助噬菌体M13K07感染后,获得重组的鸡源抗三肽囊素抗独特型抗体全套单链噬菌体抗体库,并将该抗体库展示在噬菌体表面,以利用噬菌体展示技术的强大筛选能力筛选出与三肽囊素同功能单链抗体（Bursin-ScFv）。     

Serovar identification, antimicrobial sensitivity, and virulence of Avibacterium paragallinarum isolated from chickens in Thailand

Avibacterium paragallinarum causes infectious coryza in chickens, an acute respiratory disease that has worldwide economic significance. The objectives of this study were to determine the serovars, antimicrobial resistance, and pathogenicity of A. paragallinarum isolated from chickens in Thailand. Eighteen field isolates of A. paragallinarum were confirmed by PCR. When examined by serotyping in a hemagglutination inhibition test, 10 isolates were serovar A, five isolates were serovar B, and three isolates were serovar C. The susceptibility of the isolates to 16 antimicrobial agents was tested by a disk diffusion method. All isolates were susceptible to amoxicillin-clavulanic acid. There was a high level of resistance to lincomycin and erythromycin. All isolates were resistant to cloxacillin and neomycin. A study of bacterial entry into, and survival within, chicken macrophages showed variation between isolates but no clear connection to serovar. A virulence test was performed by challenging 4-wk-old layers via the nasal route with 400 dl of bacteria (10(8) colony-forming units/ml). Clinical signs were observed daily for 7 days, and the birds were subjected to a postmortem necropsy at 7 days postchallenge. All 18 field isolates caused the typical clinical signs of infectious coryza and could be re-isolated at 7 days after challenge. There was no significant difference in the clinical scores of the isolates except that two isolates (112179 and 102984, serovars A and B, respectively) gave a significantly higher score than did isolate CMU1009 (a serovar A isolate). No correlation between serovar and severity of clinical signs was found.     

Boophilus microplus cathepsin L-like (BmCL1) cysteine protease: specificity study using a peptide phage display library

The tick Rhipicephalus (Boophilus) microplus is one of the most important bovine ectoparasites, a disease vector responsible for losses in meat and milk productions. A cysteine protease similar to cathepsin L, named BmCL1, was previously identified in R. microplus gut, suggesting a role of the enzyme in meal digestion. In this work, BmCL1 was successfully expressed in Pichia pastoris system, yielding 54.8 mg/L of culture and its activity was analyzed by synthetic substrates and against a R. microplus cysteine protease inhibitor, Bmcystatin. After rBmCl1 biochemical characterization it was used in a selection of a peptide phage library to determine rBmCL1 substrate preference. Obtained sequenced clones showed that rBmCL1 has preference for Leu or Arg at P(1) position. The preference for Leu at position P(1) and the activation of BmCL1 after a Leu amino acid residue suggest possible self activation.     

鸭坦布苏病毒HN1株囊膜E蛋白抑制肽的筛选及鉴定

鸭坦布苏病毒(DTMUV)是一个新发现的病毒,主要引起鸭产蛋严重下降为特征,为鉴定与DTMUV囊膜E蛋白特异性结合的多肽,本试验利用噬菌体展示技术以DTMUV HN1株E蛋白为靶标,进行随机12肽库3轮筛选,结合ELISA试验和竞争抑制试验,成功筛选了2个能够与DTMUV HN1株E蛋白特异性结合的多肽。测序结果显示,其氨基酸序列分别为:HWSTRQGSTRWN(P3-12)和THRSWQGNSWYM(P8-12)。进一步分析多肽P3-12和P8-12对DTMUV在鸭胚成纤维细胞增殖方面的影响,结果显示,E蛋白抑制肽P3-12和P8-12本身对鸭胚成纤维细胞的增殖无明显影响,不同质量浓度的的抑制肽P3-12和P8-12(0.02、0.2、2、20mg/L)均能显著降低DTMUV在鸭胚成纤维细胞中的病毒滴度和病毒拷贝数,以上结果表明,抑制肽P3-12和P8-12均能抑制DTMUV在鸭胚成纤维细胞的增殖,本试验为进一步探明DTMUV与宿主的相互作用及抗病毒制剂的开发提供了理论依据。     

Isolation of Lawsonia intracellularis specific single-chain Fv antibody fragments from phage display library

Single-chain antibodies (scFv) exhibiting specific binding to Lawsonia intracellularis were isolated from a phagemid library expressing scFvs molecules on the surface of filamentous bacteriophages. For scFv selection whole bacterial cells were used and individual clones were tested in ELISA test. The total of seven unique clones with different fingerprint profiles was isolated. All clones were able to bind specifically in immunofluorescence assay. This is the first report of species specific recombinant antibodies against L. intracellularis.