Cross-reactivity of monoclonal antibodies to bovine immunoglobulins with immunoglobulins of other species.

Monoclonal antibodies (Mabs) to bovine immunoglobulin heavy chain of the four major isotypes gamma 1, gamma 2, alpha, mu and the light chains (combined kappa and lambda) were produced and found to cross-react in enzyme-linked immunoassay (ELISA) with immunoglobulins of some other animal species despite the discrete specificity associated with an antibody derived from a single clone. This cross-reactivity, particularly amongst ruminants, could be utilized in serological testing for the diagnosis of disease in these species. For example, Mabs produced against bovine immunoglobulin light chain cross-react with bison immunoglobulin light chain and were used successfully in serological testing as the secondary detection antibody in an indirect ELISA for the diagnosis of Brucella abortus in bison herds in north-western Canada.     

鸡新城疫卵黄抗体IgY的分离提纯研究

本文对母鸡经鸡新城疫疫苗免疫接种后产生并富集在卵黄中的抗体IgY的提取纯化工艺条件进行了研究与优化，确定了去脂、提取抗体最佳工艺为：卵黄液用0．05mol／L,pH5.0的乙酸一乙酸钠缓冲液1：9倍稀释，搅拌15min，4℃静置10小时，抗体回收率为90％，去脂率为99％；该提取液用40％饱和度的硫酸铵盐析，得到的盐析液抗体回收率为82％，纯度达到69％。盐析液经截留分子量为100KD的有机陶瓷膜超滤后，截留液抗体回收率达到92％。纯度为85％。     

禽流感免疫抗体监测的质量控制

禽流感免疫抗体监测结果的准确性、有效性、可靠性和代表性对禽流感防控工作起着十分重要的作用,本文从样品处理、试剂配制、器材选用、温度和时间控制以及结果判定等方面对禽流感免疫抗体监测试验过程中的质量控制作-简要概述。     

PRRSV重组N蛋白的抗原性分析及其特异性抗体制备

利用大肠杆菌表达技术和亲和层析法纯化出重组PRRSV N蛋白 ,用间接ELISA法和Western blot分析 ,证明其具有较好的抗原性。将该纯化蛋白采用大剂量长程免疫法免疫家兔 ,制备了兔抗重组N蛋白抗体 ,用Western blot分析和竞争抑制试验证明其具有很高的特异性 ,ELISA效价达 1∶32 0 0以上。     

禽白血病病毒p27蛋白在大肠杆菌中的表达和多克隆抗体的制备

将禽白血病病毒p27基因克隆并构建重组表达质粒pET28a-p27,在大肠杆菌BL21中经IP TG诱导后产生可溶性表达蛋白。表达的蛋白用Western blot进行活性检测,其能与辣根过氧化物酶标记的兔抗p27抗体发生特异性反应;采用金属螯和层析对表达蛋白进行纯化,其纯度约为95％;用纯化的表达蛋白p27免疫家兔制备抗血清,ELISA抗体效价可达1∶25600。研究结果表明,大肠杆菌表达的p27蛋白具有良好的抗原性,可以替代纯化病毒蛋白用于禽白血病病毒检测。     

Clinical chemistry of companion avian species: a review

Birds have evolved alternate physiologic strategies to contend with dehydration, starvation, malnutrition, and reproduction. Basic anatomic and functional differences between birds and mammals impact clinical chemistry values and their evaluation. Interpretation of the results of standard biochemical analyses, including BUN, alanine aminotransferase, aspartate aminotransferase, creatine kinase, gamma glutamyltransferase, bilirubin, ammonia, alkaline phosphatase, cholesterol, bile acids, glucose, albumin, globulins, calcium, phosphorus, prealbumin (transthyretin), fibrinogen, iron, and ferritin, is reviewed and discussed in relation to these physiological differences. The use and interpretation of alternative analytes appropriate for avian species, such as uric acid, biliverdin, glutamate dehydrogenase, and galactose clearance, also are reviewed. Normal avian urine and appropriate use of urinalysis, an integral part of laboratory diagnosis in mammalian species that frequently is omitted from avian diagnostic protocols, is discussed.     

表达H5N1亚型禽流感HA、NA、 M1基因的重组杆状病毒的鉴定

为证实插入了禽流感HA、NA、M1、M2、NP基因的重组杆状病毒的表达物为具有禽流感形态的病毒样颗粒,对表达产物进行电镜、Western Blot、ELISA检测。蔗糖密度梯度离心纯化表达产物,电镜观察,可见禽流感病毒样颗粒;以Western Blot方法用HA、NA、M1抗体检测表达产物和纯化产物,可见特异性条带;NA多抗为捕获抗体、HA单抗为检测抗体建立夹心ELISA方法,检测表达产物,结果为阳性。结果显示,插入了禽流感HA、NA、M1、M2、NP的重组杆状病毒能够表达禽流感病毒样颗粒。     

仙游县高致病性禽流感免疫抗体监测与分析

通过对禽流感免疫抗体进行监测与分析,全面掌握禽流感的免疫效果,提高预警能力,指导养殖场(户)制定合理的免疫程序。     

免疫不同剂量禽流感疫苗对蛋鸡抗体效价的影响

禽流感作为家禽养殖业的最大威胁,通过科学使用禽流感疫苗进行免疫能够有效预防暴发禽流感疫情。本试验对蛋鸡在相同条件下注射不同剂量的禽流感疫苗,间隔相同时间后采样测定抗体的效价,结果表明:首次免疫的剂量0.3 m L/羽、0.4m L/羽、0.5 mL/羽,一个月后测定的(H5+H7)抗体滴度平均值随着免疫剂量的增加而提高,60日龄加强免疫剂量为0.5 mL/羽、0.7 mL/羽、0.9 mL/羽,一个月及两个月后测定的(H5+H7)抗体滴度平均值均保持在最高水平10。试验表明:首次免疫禽流感疫苗剂量为0.5 m L/羽的抗体效价水平最高,加强免疫的不同剂量均能使抗体效价在较长一段时期保持最高水平。     

A survey of avian Mycoplasma species for neuraminidase enzymatic activity

Among 23 currently recognized avian Mycoplasma (AM) species only Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma meleagridis and Mycoplasma iowae cause disease and loss of production in chickens and/or turkeys. Because neuraminidases are considered virulence factors in many pathogenic microorganisms the aim of our study was to determine which AM species possess neuraminidase enzymatic activity (NEAC). Small samples of AM cells were assayed for NEAC using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-alpha-d-N-acetylneuraminic acid. In the case of positive NEAC reaction the substrate gave the insoluble indigoblue product what enabled simple test and easy estimation of NEAC. M. gallisepticum and M. synoviae which share sequences of the gene encoding neuraminidase (sialidase NanH) exhibited considerable levels of NEAC. However, NEAC levels differed among their strains, as well as among cultures of different strains. Only certain cultures of the type strain of M. meleagridis showed NEAC, whereas among six serovars of M. iowae only serovar I (type strain 695) showed NEAC. Weak NEAC was detectable in M. anseris, M. cloacale and M. pullorum, whereas the type strain of M. corogypsi (BV1) showed strong NEAC. Our study provides novel informations about NEAC in AM species and suggests that higher invasiveness and possibly, the pathological processes might be associated with their NEAC.     

H9亚型禽流感病毒基质蛋白M1单克隆抗体的制备与鉴定

本研究用纯化的H9N2亚型禽流感病毒免疫BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合。对杂交瘤细胞及时筛选,阳性孔经3次有限稀释法克隆,成功获得3株能稳定传代并分泌抗H9亚型禽流感病毒基质蛋白M1单克隆抗体的杂交瘤细胞：3G8、2F6、5F2。间接ELISA方法检测,3株单克隆抗体的腹水间接ELISA效价达106以上。构建了真核表达载体pCAGGS-M1并转染于MDCK细胞,以用于腹水的Western blot和间接免疫荧光鉴定。间接免疫荧光结果表明,3株单克隆抗体皆与真核表达蛋白M1反应。这些单克隆抗体的制备为后期研究M1蛋白在流感病毒复制与出芽过程中的重要生物学功能奠定了基础。     

The distribution and heterogeneity of mast cells in tongue from five different avian species

This study was conducted with the aim of determining the morphology, distribution and heterogeneity of mast cells in the tongues of seagull (Larus fuscus), common buzzard (Buteo buteo), goose (Anser anser), white stork (Ciconia ciconia) and Gerze rooster. The study used five samples of tongue material from each of the healthy adult avian species. The samples were fixed in 10% neutral‐buffered formalin (NBF) solution, then, after routine tissue follow‐up, the samples blocked with paraplast. Cross‐sections with 5–6 μm of thickness were stained with the 0.5% toluidine blue and alcian blue/safranin O (AB/SO). In all five avian species, it was found that the mast cells were in different sizes and round, oval or spindle‐shaped based on their place of distribution. Mast cell numbers were determined in stained with toluidine blue, examined ×40 objectives in a 1 mm² area. It was observed that mast cell density in subepithelial lamina propria and microscopic papilla was higher in the tongues of all species. Mast cell distribution and heterogeneity varied through the tongue, and there were more mast cells in the dorsal side of the tongue than the ventral side. The highest amount of mast cells was found in the tongue of the Gerze rooster among all five species. In the tongue cross‐sections stained with the combined method of alcian blue/safranin O (AB/SO), the mast cells were stained as AB (+), SO (+) and AB/SO (+) (mixed).     

重组N蛋白抗原检测PRRSV抗体ELISA的研究Ⅳ.与IDEXX ELISA试剂盒及Western blot的比较

利用重组N蛋白抗原建立了检测猪繁殖与呼吸综合征病毒（PRRSV）抗体的ELISA方法并组装成试剂盒，将试制的3批试剂盒分别与IDEXX生产的试剂盒及Western—blot进行了符合率试验。试验结果表明，所研制的试剂盒与Western blot的符合率达97．67％以上。对460份临床血清分别用自制的试剂盒和IDEXX公司生产的试剂盒进行检测，其中有37份不相符合，用Western blot对这37份血清进行验证，有35份血清检测结果与自制的试刺盒检测结果一致。由此表明，自行研制的试剂盒，其特异性和敏感性均能满足目前临床上该疫病的流行病学分析或免疫抗体检测。     

含有鸡体偏嗜性密码子的H5亚型禽流感病毒HA基因的优化构建及其DNA疫苗体外瞬时表达研究

不同物种间细胞在蛋白翻译过程中具有密码子使用的偏嗜性,鸡与A型流感病毒的密码子使用频率存在着明显差异.为此,利用寡核苷酸合成、重叠延伸PCR及限制酶切法构建了密码子优化的表达A/Goose/GuangDong/1/96(H5N1)[GD/1/96(H5N1)]HA蛋白的基因optiHA5,并将其插入CMV启动子表达载体pCI构建了H5亚型DNA疫苗质粒pCIoptiHA5,将含有GD/1/96(H5N1)野生型HA基因的质粒pCIHA5和pCIoptiHA5分别转染293T细胞,间接免疫荧光检测转染后24～48h293T细胞中瞬时表达的HA抗原蛋白;Westemblot证实上述2种表达质粒均可正确表达H5亚型HA抗原蛋白,结果表明密码子优化的基因optiHA5体外瞬时表达水平显著高于野生型HA基因.这一结果为进一步开展SPF鸡免疫保护效果的比较研究奠定了基础.     

大鼠成熟的肺表面活性蛋白B的原核表达及纯化

为表达和纯化SP-B蛋白,先对SP-B基因进行稀有密码子优化,PCR反应获得SP-B片段,构建重组质粒pGEX4T-1/SP-B,转化到E.coli BL21(DE3)中诱导表达;用SDS-PAGE与Western blot进行检测;用GSTPrep FF 16/10对融合蛋白进行纯化。经双酶切鉴定证实质粒中插入基因长250bp,测序结果与大鼠SP-B cDNA序列相符;质粒转化后用IPTG进行诱导,在分子质量约34ku处出现1个新条带,与pGEX4T-1/SP-B蛋白预期大小一致,且该融合蛋白能可溶性表达并被纯化。结果表明成功构建了pGEX4T-1/SP-B重组质粒,表达、纯化得到GST/SP-B蛋白,为研究肺表面活性物质替代药物奠定了基础。     

禽流感病毒NP基因的真核表达及抗体检测芯片的建立

为建立一种检测A型禽流感病毒(AIV)抗体的蛋白芯片,本研究以AIV总RNA为模板,RT-PCR方法扩增NP基因,与真核表达载体pFastBacHTa连接,并在昆虫细胞中表达.重组蛋白经纯化及western blot鉴定,点样于醛基包被的载玻片上,制备检测AIV抗体的蛋白芯片.确定芯片反应最佳条件为:点样浓度2 mg/mL,固定24 h,1%BSA进行封闭,一抗稀释度1:100,二抗稀释度1:2000.利用蛋白芯片分别对15个亚型流感病毒免疫血清、SPF鸡血清、抗新城疫病毒血清、抗法氏囊病毒血清和现地血清样品进行检测,通过与血凝抑制试验比较,表明建立的蛋白芯片方法具有良好的灵敏性和特异性,为AIV抗体检测及制备检测AIV不同亚型或多种病原抗体的蛋白芯片提供方法.     

血管瘤禽白血病病毒FJ0610株的分离与鉴定

为了解禽白血病病毒在商品蛋鸡中的流行情况,试验采用病理剖检、聚合酶链式反应(PCR)以及间接免疫荧光试验(IFA)等方法对送检的疑似禽白血病病毒感染的商品蛋鸡进行了病毒分离与鉴定,并对分离株的致瘤相关基因gp85基因进行测序,与国内外各亚群禽白血病病毒进行对比。结果表明:分离、鉴定到1株J亚群血管瘤禽白血病病毒,命名为FJ0610;分离株gp85基因核苷酸序列同源性在11.5%～94.5%之间,其中与血管瘤禽白血病毒株ZH-08株同源性最高,而与E亚群毒株同源性最低;基于gp85核苷酸序列的系统进化分析表明FJ0610株的gp85序列与ZH-08株的亲缘关系最近。     

2019年秋季双鸭山市禽流感和鸡新城疫集中抗体监测与分析

通过血凝和血凝抑制试验方法,对双鸭山市内各区秋季集中免疫禽流感和新城疫的鸡群进行免疫监测,结果显示,禽流感H5亚型抗体监测群体合格率为94.3%,禽流感H7亚型抗体监测群体合格率为95.2%,鸡新城疫抗体监测群体合格率为90.4%。参照农业部相关规定,灭活疫苗家禽免疫21 d后监测群体合格率要求≥70%,结果表明,秋季集中免疫禽流感和鸡新城疫抗体水平较高,具有很好的保护力。     

Use of personal protective measures by Thai households in areas with avian influenza outbreaks

Thailand has had multiple poultry outbreaks of highly pathogenic avian influenza (HPAI) H5N1 since its first emergence in 2004. Twenty-five human cases of HPAI H5N1 avian influenza have been reported in the country, including 17 fatalities, and contact with infected dead or dying poultry has been identified as a risk factor for human infection. This study assessed the use of protective equipment and hand hygiene measures by Thai poultry-owning households during activities involving poultry contact. Surveys conducted in 2008 included questions regarding poultry-related activities and protective measures used during an HPAI outbreak (2005) and 3 years after the study location's last reported outbreak (2008). For both time periods, poultry owners reported limited use of personal protective equipment (PPE) during all activities and inconsistent hand washing practices after carrying poultry and gathering eggs. This is the first time that PPE use in Thailand has been quantified for a large study group. These data are important for ongoing characterization of HPAI risk and for the crafting of educational messages.