禽脑脊髓炎病毒单克隆抗体的制备、鉴定与初步应用

为获得禽脑脊髓炎病毒(Avian Encephalomyelitis virus,AEV)VP1蛋白的单克隆抗体,通过原核表达AEV VP1蛋白,纯化后作为免疫原免疫BALB/c小鼠,并按常规方法制备杂交瘤细胞。经ELISA方法筛选阳性杂交瘤细胞,经过3次亚克隆获得2株杂交瘤细胞株,命名为4#、19#,并进行了抗体亚类的鉴定、Western-blot和IFA检测。结果显示：制备的株单克隆抗体亚型分别为IgG2b、IgG2a,Western-blot和IFA试验结果表明单克隆抗体均能与AEV发生特异性反应而与其他禽病常见病毒均无交叉反应。运用建立的IFA对单抗进行了初步运用,在外源病毒检测方面与经典方法符合率高。本研究成功制备了AEV单克隆抗体,为进一步建立AEV检测方法和深入研究AEV的生物学特性奠定了基础。     

鸡传染性支气管炎病毒特异性单克隆抗体的制备与鉴定

为获得鸡传染性支气管炎病毒(Avian infectious bronchitis virus, IBV)N蛋白的单克隆抗体,通过原核表达IBV N蛋白,纯化后作为免疫原免疫BALB/c小鼠,并按常规方法制备杂交瘤细胞。经ELISA方法筛选阳性杂交瘤细胞,经过3次亚克隆获得3株杂交瘤细胞株,命名为1#、18#、19#,并进行了抗体亚类的鉴定、Western-blot和IFA检测。结果显示：制备的3株单克隆抗体亚型均为IgG1,Western-blot和IFA试验结果表明单克隆抗体均能与IBV发生特异性反应而与其他禽病常见病毒均无交叉反应。本研究成功制备了IBV单克隆抗体,为进一步建立IBV检测方法和深入研究IBV的生物学特性奠定了基础。     

Detection of specific antibody responses to vaccination in variable flying foxes (Pteropus hypomelanus)

Megachiropteran bats are biologically important both as endangered species and reservoirs for emerging human pathogens. Reliable detection of antibodies to specific pathogens in bats is thus epidemiologically critical. Eight variable flying foxes (Pteropus hypomelanus) were immunized with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA). Each bat received monthly inoculations for 2 months. Affinity-purified IgG was used for production of polyclonal and monoclonal anti-variable flying fox IgG antibodies. ELISA and western blot analysis were used to monitor immune responses and for assessment of polyclonal and monoclonal antibody species cross-reactivity. Protein G, polyclonal antibodies, and monoclonal antibodies detected specific anti-DNP antibody responses in immunized variable flying foxes, with protein G being the most sensitive, followed by monoclonal antibodies and then polyclonal antibodies. While the polyclonal antibody was found to cross-react well against IgG of all bat species tested, some non-specific background was observed. The monoclonal antibody was found to cross-react well against IgG of six other species in the genus Pteropus and to cross-react less strongly against IgG from Eidolon helvum or Phyllostomus hastatus. Protein G distinguished best between vaccinated and unvaccinated bats, and these results validate the use of protein G for detection of bat IgG. Monoclonal antibodies developed in this study recognized immunoglobulins from other members of the genus Pteropus well, and may be useful in applications where specific detection of Pteropus IgG is needed.     

Paramyxovirus type 1 infections of racing pigeons: 1 characterisation of isolated viruses

Viruses isolated from field outbreaks of disease in racing pigeons in continental Europe and Great Britain were shown to be identical by serological tests using conventional chicken antisera and mouse monoclonal antibodies. The pigeon viruses showed high levels of cross-reaction to Newcastle disease virus (NDV) in haemagglutination inhibition tests and Madin-Darby bovine kidney cells infected with pigeon virus isolates bound three out of nine mouse monoclonal antibodies prepared against NDV Ulster 2C. These results confirm their classification in the paramyxovirus type 1 serotype of avian paramyxoviruses. However, the pigeon viruses could be distinguished from more classical paramyxovirus type 1 viruses by the significantly different titres obtained in haemagglutination inhibition tests, the failure of mouse monoclonal antibodies directed against the HN1 epitope of NDV Ulster 2C to inhibit their haemagglutinating activity and a unique binding pattern seen with the nine mouse monoclonal antibodies.     

An improved radioimmunoassay for the anabolic agent hexoestrol, using a monoclonal antibody

A monoclonal antibody was raised against hexoestrol coupled to bovine serum albumin. The antibody cross-reacted with the stilbenes, diethylstilboestrol (10%) and dienoestrol (4%), but had no cross-reaction (less than 0.01%) with other anabolic agents. A radioimmunoassay method using the monoclonal antibody has been validated and used to measure residues of hexoestrol in the urine of treated cattle. The limit of detection was 0.6 pg/ml urine at the 95% confidence limit. The results were compared with those obtained using polyclonal antibodies. Although there was a good correlation between the results, the use of monoclonal antibody gave more reliable results than those obtained with available polyclonal antibodies. The monoclonal antibody, because of its quality and theoretically limitless supply, is very suitable for use in large scale screening or monitoring programmes for regulating the use of hexoestrol.     

Characterization of monoclonal antibodies against ovine Sarcocystis spp. antigens by immunoblotting and immuno-electron microscopy

Six monoclonal antibodies were raised in mice against purified cytozoite extracts of Sarcocystis gigantea and S. tenella from sheep. Each monoclonal antibody was evaluated for specificity by enzyme immunoassay, immunoblotting and immuno-electron microscopy using homologous and heterologous antigenic preparations. All six monoclonal antibodies exhibited good species-specificity when reacted against crude soluble cystozoite antigens in enzyme immunoassays. However, only two monoclonal antibodies (IgM and IgG2a) exhibited reactivity in Western blots against specific protein bands. Both reacted against S. gigantea antigens of 100,000, 43,000 and 39,000 molecular weight. Neither monoclonal antibody reacted against the heterologous species S. tenella. Ultrastructural studies performed with colloidal-gold conjugated antisera revealed that both monoclonal antibodies reacted against antigens located around micronemes and amylopectin granules in S. gigantea cystozoites. Another monoclonal antibody (IgGI) reacted only against microneme determinants in S. tenella cystozoites. In contrast, polyclonal sheep and rabbit immune sera cross-reacted against a wide range of cystozoite antigens.     

Amyloid in the horse: a report of nine cases

Out of approximately 16,000 horses referred for clinical examination, nine had amyloidosis. Six of these horses had localised amyloid deposits in the wall of the nasal meatus and ventral turbinates associated with epistaxis. Horse 1 also developed malignant histiolymphocytic lymphosarcomas. The amyloid deposits were potassium permanganate-resistant and tryptophan-positive. Gel filtration of solubilised amyloid fibrils from Horse 1 revealed a major retarded fraction with an apparent molecular weight of 20 kD. This protein had an amino acid composition similar to human AL-amyloid proteins and horse immunoglobulin light chains. On Western blot a strong cross-reaction was observed between horse 1gG2a light chains and the Horse 1 amyloid. Horses 7 to 9 had suppurative verminous aneurysm, tuberculosis and an adrenal cortical adenoma, respectively, and had generalised amyloid deposits in liver and spleen. These amyloid deposits were found to be potassium permanganate-sensitive and positive for tryptophan. Gel filtration of solubilised amyloid fibrils from Horse 8 revealed a major retarded fraction (protein AA) with an apparent molecular weight of 10 kD. Immunoperoxidase-antiperoxidase staining showed the localised deposits to be negative or only weakly positive with antisera against bovine, hamster, dog and human protein AA and to be positive with anti-horse-one amyloid protein. The generalised deposits were found to be positive with the antisera against allogenic protein AA. The results of the potassium permanganate incubation, biochemistry, immunoblotting and immunochemistry, indicate that the localised amyloid of Horse 1 and most likely the amyloid of Horses 2 to 6, is of the AL-type. The generalised amyloid deposits were found to be of the AA type.     

The sustainability, feasibility and desirability of breeding livestock for disease resistance

The sequence encoding a truncated E2 glycoprotein of the Alfort/187 strain of classical swine fever virus (CSFV) was expressed in Escherichia coli using the pET expression system and the recombinant product purified by Ni-NTA agarose affinity chromatography. The antigenicity of this recombinant protein was demonstrated by immunoblot using anti- CSFV-specific antibodies. A monoclonal antibody was produced against the truncated E2 protein and used as competitor in an ELISA for the detection of antibodies to CSFV. Specific antibodies were demonstrated by competitive ELISA (C-ELISA) as early as 21 days post-infection (dpi) in experimentally infected pigs. Seroconversion was demonstrated by C-ELISA and neutralising peroxidase-linked assay (NPLA) in all infected animals by 4 weeks. No cross-reaction with antibodies to bovine viral diarrhoea virus (BVDV) was seen in the C-ELISA using sera from experimentally infected pigs. The C-ELISA is not intended as a substitute for the NPLA. However, it is expected it will be useful for monitoring and prevalence studies. It will also assist in testing a large number of samples in the event of an outbreak.     

伪狂犬病病毒闽A株单克隆抗体的制备与鉴定

本试验应用细胞杂交瘤技术,取健康BALB/c小鼠脾细胞与SP2/0 骨髓瘤细胞进行细胞融合,经双抗体夹心ELISA筛选,4次有限稀释法克隆,得到2株能稳定分泌抗伪狂犬病病毒(PRV)的单克隆抗体杂交瘤细胞株:2C6E6、3D5F1。2株杂交瘤细胞培养上清的效价分别为1:512、1:1 024。鉴定结果显示这2株单克隆抗体分别为IgG1亚类和IgG2a亚类,杂交瘤细胞的平均染色体数目为84条。用纯化的PRV免疫经产健康的BALB/c小鼠,采用ELISA方法检测获得的腹水的效价分别为1:1 638 400和1:819 200。经检测这2株单克隆抗体与猪瘟病毒、猪繁殖与呼吸综合征病毒、猪细小病毒均不发生交叉反应,特异性良好。杂交瘤细胞连续培养30代,仍能稳定分泌抗PRV的单克隆抗体,说明这2株单克隆抗体的稳定性良好。本试验研究结果为PRV的快速诊断方法的建立奠定了理论基础。     

Preparation and Identification of Monoclonal Antibody against Min-A Strain of Pseudorabies Virus (PRV)

With purified pseudorabies virus (PRV) to immune BALB/c mice, spleen cells from imunized mice were fused with SP2/0 myeloma cells by application of lymphocyte hybridoma technique, followed by double antibody sandwich ELISA screening, four times cloning by limiting dilution method to obtain two stable secreting anti-PRV monoclonal antibody hybridoma cell lines, 2C6E6 and 3D5F1.After identification, these two monoclonal antibodies belonged to IgG1 and IgG2a subtypes, respectively, the average number of chromosomes of hybridoma cells was 84, ELISA titers of these two hybridoma cell culture supernatants and mouse ascites monoclonal antibodies were 1:512 and 1:1 638 400, 1:1 024 and 1:819 200.These two monoclonal antibodies showed no cross-reaction with classical swine fever virus, porcine reproductive and respiratory syhdrome virus and porcine parvovirus, which indicated that they had high specificity.The hydridoma cells could passage for 30 generations which could still secrete monoclonal antibody against PRV, which indicated that these two monoclonal antibodies had good stability.The results in the assay laid the foundation for establishing rapid diagnostic methods of PRV.     

非洲猪瘟病毒DP96R蛋白的表达与单克隆抗体的制备

为制备非洲猪瘟病毒(ASFV) DP96R蛋白单克隆抗体,根据大肠杆菌密码子优化后的DP96R基因序列设计引物,PCR扩增后连接表达载体pET28a-SUMO构建pET-SUMO-DP96R原核表达质粒,将该质粒转化大肠杆菌BL21细胞,经IPTG诱导,获得可溶性的DP96R蛋白.通过Western blot鉴定该蛋白...     

Ovicidal and larvicidal activity in vitro of Spigelia anthelmia Linn. extracts on Haemonchus contortus

The prevalence of antibodies to Neospora caninum was examined in six wild Artiodactyla species, and in five wild Carnivora species from Kenya. Blood sera (104 wild ungulates from Marula Estates (MEs), and 31 wild carnivores from Masai-Mara reserve and from other wildlife areas in northern and Southern Kenya), were screened using a Neospora agglutination test (NAT), with a twofold dilution (1:40-1:320 titres). Presence of NAT antibodies to N. caninun is reported here for the first time in zebra (Equus burchelli), eland (Taurotragus oryx), African buffalo (Syncerus caffer), Thompson gazelle (Gazella thompsoni), impala (Aepyceros melampus), warthog (Phacochoerus aethiopicus), spotted hyena (Crocuta crocuta) and in free-ranging cheetah (Acinonyx jubatus). At 1:80 dilution, prevalence was 61.5% in eland, 58.5% in zebra, 19.2% in Thompson gazelle, 33.3% in warthog, 50% in African buffalo, 30% in lion (Panthera leo), 20% in cheetah, and 33.3% in spotted hyena. Antibodies up to 1:320 titre were detected in eland (38.4%), zebra (19.5%), Thompson gazelle (3.8%) and lion (5%). Amongst herbivores, sero-prevalence was significantly (P<0.05) higher, at all dilutions, in "grazer/digger" species (e.g. eland and zebra) than in non-"grazer/digger" species (e.g. impala and Thompson gazelle). No antibodies to N. caninum were found in two leopards (Panthera pardus) and one serval (Felis serval). Our results indicates a steady presence of N. caninum in wild mammals from Kenya. The hypothesis of a sylvatic cycle of N. caninum could be suggested, but more data are needed to verify the hypothesis, as to evaluate the role of N. caninum infection on the dynamics of wild animals population in the study area.     

牛朊蛋白和酵母朊毒体结构域融合蛋白的原核表达及其单克隆抗体的制备

利用DNA重组技术将酵母Ure2p朊蛋白结构域(UPD)基因插入牛朊蛋白(BPrP)表达质粒pET-PrP中,构建原核表达载体pET-PrP-UPD.阳性质粒转化宿主菌BL21 (DE3),在IPTG诱导下获得高效表达.Westernblot检测表明表达的重组蛋白与单克隆抗体6H4呈现特异性反应,且纯化的PrP-UPD融合蛋白在体外具有聚集成淀粉样纤维、抵抗蛋白酶K消化等朊毒体的结构特点.利用纯化的PrP-UPD免疫BALB/c小鼠,取其脾细胞与SP2/0细胞融合,经克隆和筛选,获得3株稳定分泌抗重组PrP单抗的杂交瘤细胞,分别命名为1G3、3A4、4D1,其中1G3分泌的单抗能识别细胞型牛朊蛋白,其Ig亚类为IgG2b,腹水ELISA效价为1×105,Western blot表明该单抗具有较强的特异性.     

Leukocyte responses in two breeds of layer chicken that differ in susceptibility to induced amyloid arthropathy

Amyloid arthropathy in chicken can be induced by intravenous inoculation of an arthropathic and amyloidogenic Enterococcus faecalis in susceptible breeds. The commercial brown layer hybrids (BL) are more susceptible to the disease compared to their white counterparts (WL). The precursor of amyloid-A protein, which is serum amyloid-A (SAA), is identical in WL and BL. To investigate the factors involved in the breed-restricted susceptibility to amyloid arthropathy, we studied the type of leukocyte response and inflammatory reactions in E. faecalis-induced disease. In the BL, a significant dose dependent peripheral leukocytosis mainly by heterophils, and plasma cell infiltration in arthritic joints was found. In contrast, secondary lymphoid nodular aggregates in the synovial membrane were prominent in the WL. The aggregates consisted mainly of CD8+ T cells. The high number of circulating leukocyte and prolific plasma cell responses in the BL predict extensive humoral and acute phase reactions. This is in agreement with literature data on suppressed T-cell function in casein-induced amyloid-susceptible mice strains. The difference in leukocyte response and type of inflammation between WL and BL, when arthropathic and amyloidogenic bacteria induce infection, in conjunction with susceptibility to amyloid arthropathy, is discussed in view of the murine T-helper responses.     

绿色荧光蛋白的真核表达及其单克隆抗体制备

【目的】构建表达绿色荧光蛋白(green fluorescent protein, GFP)的重组猪痘病毒(recombinant Swinepox virus, rSWPV),制备抗GFP单克隆抗体。【方法】首先合成3′-端含His标签的增强型绿色荧光蛋白(EGFP)序列EGFP-His,用双酶切方法将其插入到基础质粒载体pSW中,构建重组转移载体pSW-EGFP-His;采用脂质体转染的方法使该载体与SWPV(SWPV-JX20G株)同源重组,经蚀斑纯化获得rSWPV-EGFP-His。重组病毒进行PCR和SDS-PAGE鉴定,扩增并纯化EGFP-His蛋白免疫BALB/c小鼠,将小鼠脾细胞与SP2/0细胞融合,筛选分泌抗GFP特异性抗体的杂交瘤细胞,制备腹水,对抗GFP单克隆抗体进行效价及特异性鉴定。【结果】PCR和SDS-PAGE结果显示,成功构建并纯化到rSWPV-EGFP-His,该病毒感染PK15细胞可稳定表达EGFP-His蛋白,蛋白大小约27 ku,为可溶性表达,镍柱纯化的EGFP-His蛋白溶液呈明显的绿色。EGFP-His蛋白免疫BALB/c小鼠,免疫小鼠血清抗体...     

The efficacy of ivermectin and levamisole against natural Nematodirus spathiger infection in the Arabian sand gazelle (Gazella subgutturosa marica) and the Arabian mountain gazelle (Gazella gazella) in Saudi Arabia

The efficacy of two broad spectrum anthelmintics (Ivermectin and Levamisole) against the nematode Nematodirus spathiger in the reem (Arabian sand gazelle) and idmi (Arabian mountain gazelle) at King Khalid Wildlife Research Centre (KKWRC) was determined. Gazelles of both species naturally infected with N. spathiger were each divided into 3 groups. One group was treated with Ivermectin, the other group with Levamisole and the last group was left as untreated control. Faecal egg counts were employed to determine the efficacy of the two drugs against N. spathiger infection in both gazelle species. Reduction in arithmetic mean faecal egg counts achieved by Ivermectin and Levamisole in the reem gazelles was 94% and 89.3%, respectively. The reduction in the idmi gazelles was 97.2% and 96.4%, respectively. Hence, both anthelmintics appeared to be more effective in the idmi gazelles than in the reem gazelles, however, there was no significant difference in the faecal egg reduction tests in both animal species.     

禽腺病毒血清4型Fiber2蛋白单克隆抗体制备及其抗原表位鉴定

旨在制备禽腺病毒血清4型（FAdV-4）纤突蛋白（Fiber2）的特异性单克隆抗体（MAb）,本研究将原核表达的可溶性重组蛋白NusA-Fiber2作为免疫原免疫BALB/c雌鼠,筛选获得3株能稳定分泌抗FAdV-4 Fiber2蛋白MAb的杂交瘤细胞株2G5、2G8、4C2,取细胞株2G5制备腹水并纯化,利用间接免疫荧光试验（immunofluorescence assay,IFA）和Western blot鉴定该单抗的特异性。用制备的单抗包被酶标板,通过一系列的优化,建立了FAdV-4 Fiber2双抗夹心ELISA检测方法。通过逐步截短分析鉴定出单克隆抗体识别的抗原表位区域。结果表明：成功获取3株单克隆细胞株2G5、2G8、4C2。MAb 2G5可与原核表达纯化的Fiber2蛋白及FAdV-4特异性反应。建立的双抗夹心ELISA检测方法具有很好的特异性、灵敏性和重复性。该MAb识别的表位序列是N端aa1—33。本研究成功制备了具有良好Western blot和IFA反应原性的单克隆抗体,为Fiber2蛋白功能研究和FAdV-4新型表位疫苗商品化研发奠定了基础。     

A competitive ELISA using anti-N monoclonal antibodies for specific detection of rinderpest antibodies in cattle and small ruminants.

A competitive ELISA (C-ELISA) using monoclonal antibodies (mAbs) which bind to the nucleo-protein (NP) of rinderpest virus (RPV) for detection of RPV antibodies in cattle and small ruminant sera is described. Unlike virus neutralisation test (VNT), this test using mAb IVB2-4, can detect specific RPV antibodies without showing a cross-reaction with antibodies to peste-des-petits ruminants-virus (PPRV); by contrast, when mAb VE4-1 is used the test detects both RPV and PPRV antibodies, including low levels of antibodies that can be found in sera containing maternal antibodies. Although antibodies to the PPRV 75-1 strain are also detected with mAb 51-5-6, the test is suitable for assessing the immune status of cattle against the Rinderpest Old Kabete (RBOK) strain. The results from a panel of sera with a known status of vaccination provide evidence for a highly significant correlation between C-ELISA and VNT. This test may be a useful tool for a standardized and accurate determination of the immunity status of both cattle and small ruminants.     

虾源副溶血性弧菌PirB蛋白单克隆抗体的制备及鉴定

用杆状病毒表达系统表达虾源副溶血性弧菌编码蛋白PirB的重组蛋白,用SDS-PAGE、Western blot对其进行鉴定,纯化后的PirB重组蛋白免疫Balb/c小鼠后进行细胞融合,通过间接ELISA方法筛选阳性杂交瘤细胞,用ELISA方法对单克隆抗体进行测定。结果发现,共筛选到7株能够稳定分泌抗PirB蛋白特异性单克隆抗体的杂交瘤细胞,小鼠腹水的效价最高效价达到1∶2.56×106,制备的单克隆抗体可以特异性识别PirB蛋白,表明成功制备了虾源副溶血性弧菌PirB蛋白单克隆抗体,为虾源副溶血性弧菌引发的疾病诊断奠定了基础。     

水泡性口炎病毒双抗体夹心ELISA检测方法的建立

为建立方便快捷的水泡性口炎病毒(VSV)检测方法,本研究以抗VSV单克隆抗体(MAb)为捕获抗体,兔抗VSV多克隆抗体为检测抗体,建立VSV双抗体夹心ELISA检测方法。结果显示,该方法的最佳工作条件为:抗VSV MAb 1A2的包被浓度为3.09μg/mL,兔抗VSV多克隆抗体和酶标抗体的工作浓度分别为5.16μg/mL和1∶5 000,以OD450nm≥0.231作为阳性判定标准。该ELISA方法对猪水泡病病毒、猪水疱疹病毒及羊传染性脓疱病毒等均无交叉反应;敏感度可达3.125μg/mL(101TCID50);其重复性变异系数小于10%。采用建立的ELISA方法与RT-PCR方法同时检测187份临床样品,符合率达到97.9%,具有良好的相关性。本实验建立的VSV双抗体夹心ELISA检测方法具有特异性好、敏感性高、成本低及方便快捷等优点,可以用于VSV的快速检测。