用单抗介导的荧光抗体技术检测鸡尿囊细胞内传染性支气管炎病毒的研究

用传染性支气管炎病毒（ＩＢＶ）Ｈ１２０、Ｈ５２、Ｍ４１、ＡＲＫ、澳大利亚Ｔ株、野外分离株ＲＳ、ＲＹ及鸡新城疫病毒（ＮＤＶ）、鸡传染性法氏囊病毒（ＩＢＤＶ），鸡传染性喉气管炎病毒（ＩＬＴＶ）、减蛋综合症病毒（ＥＤＳＶ）等分别接种９日龄ＳＰＦ鸡胚，３７℃孵育４８小时后，将其尿囊液离心，沉淀用ＰＢＳ悬浮，涂片，用抗ＩＢＶ单抗ＭＣ作一抗、进行间接荧光抗体检测。结果：Ｈ１２Ｏ、Ｈ５２、Ｍ４１“ＡＲＫ、Ｔ株及ＲＳ、ＲＹ均显阳性，而ＮＤＶ、ＩＢＤＶ、ＩＬＴＶ、ＥＤＳＶ均为阴性。结果表明、用此法检测鸡胚尿囊液中ＩＢＶ．具有快速、敏感、特异的优点。     

分泌抗禽呼肠孤病毒单克隆抗体杂交瘤细胞株的建立与鉴定

采用腹腔的接种１次脾内注射禽呼肠孤病毒（ＡＲＶ）蛋白免疫小鼠，经３次融合后，共筛选８株分泌抗禽呼肠孤病毒（单克隆抗体杂交瘤细胞株，这８株ＭｃＡｂ均可与ＡＲＶＳ１１３３株，ＦＤＯ株发生反应，而与ＩＢＤＶ，ＭＤＶ，ＥＤＳ－７６病毒不发生反应，经亚类鉴定，ＡＥ７，ＡＦ８，ＢＤ１，ＤＨ１０，ＥＥ５为ＩｇＧ１；ＡＤ６，ＣＧ４为ＩｇＣ２ａ，ＡＧ７为ＩｇＧ２ｂ。腹水效价在１０＾３～１０＾５之间。     

鸡毒支原体株间结构蛋白及其抗原性变异的比较研究

本研究以ＳＤＳ－ＰＡＧＥ及Ｗｅｓｔｅｒｎ Ｂｌｏｔ技术,应用鸡毒支原体国外强毒株Ｓ６、标准株ＰＧ３１,疫苗株Ｆ,北京分离 ＢＧ４４Ｔ、ＮＢ７２特异性多克隆抗血清对以上五株及疫苗株Ｖ、北京分离株Ｃ的结构及其抗原性进行了比较结果表明ＭＧ结构蛋白及其抗原性存在着株间的多样性和一定相似性,其中以Ｆ与Ｓ６、ＢＧ４４Ｔ与ＰＧ３１、ＮＢ７２与Ｃ更为接近,可能具有同源性。ＳＤＳ－ＰＡＧＥ显示出了ＭＧ株间结构蛋白微     

马立克氏病病毒囊膜糖蛋白B抗原Ⅰ型特异性和群共同性决定簇的单克隆抗体

用能表达马立克氏病病毒（ＭＤＶ）糖蛋白Ｂ（ｇＢ）的重组杆状病毒感染的Ｓｆ９细胞免疫小鼠，制备针对ＭＤＶｇＢ的单克隆抗体。以Ⅰ型马立克氏病病毒ＧＡ株感染的鸡胚成纤维细胞作为检测抗原，同时以免疫荧光试验（ＩＦＡ）和酶联免疫吸附试验（ＥＬＩＳＡ）来筛选杂交瘤细胞，结果获得了ＩＦＡ和ＥＬＩＳＡ均为阳性的２株单克隆抗体细胞株，定名为ＢＡ４和ＢＤ８。在ＩＦＡ和免疫沉淀试验中，单抗ＢＤ８与Ⅰ、Ⅱ、Ⅲ型ＭＤＶ均呈阳性反应；单抗ＢＡ４只对Ⅰ型ＭＤＶ（包括ＣＶＩ９８８疫苗株）呈阳性反应。免疫沉淀反应进一步确证２株单抗识别的是ＭＤＶ糖蛋白Ｂ抗原。     

用单抗介导的荧光抗体技术检测鸡尿囊细胞内传染性支气管炎病毒的研究

用传染性支气管炎病毒Ｈ１２０、Ｈ５２、Ｍ４１、ＡＲＫ、澳大利亚Ｔ株、野外分离株ＲＳ、ＲＹ及鸡新城疫病毒（ＮＤＶ）、鸡传染性法氏囊病毒、鸡传染性喉气管炎病毒，减蛋综合症病毒等分别接种９日龄ＳＰＦ鸡胚，３７Ｃ孵育４８小时后，将其尿囊液离心，沉淀用ＰＢＳ悬浮，涂片，用抗ＩＢＶ单抗ＭＣ作一抗，进行间接荧光抗体检测。结果：Ｈ１２０、Ｈ５２、Ｍ４１“ＡＲＫ、Ｔ株及ＲＳ、ＲＹ均显阳性，而ＮＤＶ、ＴＢＤＶ、ＩＬＴ     

鸽新城疫病毒的分离及其生物学特性测定

用ＳＰＦ鸡胚从疑似鸽新城疫（鸽ＮＤ）病鸽群中分离到一株病毒ＱＬ株,该病毒株能凝集鸡红细胞（ＲＢＣ）,这种凝集作用能被抗新城疫病毒（ＮＤＶ）阳性血清抑制;用抗ＮＤＶ单抗ＰＥＧ夹心ＥＬＩＳＡ测定分离株为阳性。分离株经肌肉注射能使鸽发病和死亡,出现与自然发病鸽一致的症状和病变,但肌注ＳＰＦ鸡只感染,不见临床症状。对该分离株作进一步生物学特性鉴定,按照国际上规定的ＮＤＶ毒力判定标准,测定了该毒株最低致死量致死鸡胚的平均死亡时间（ＭＤＴ）、１日龄雏鸡脑内接种致病指数（ＩＣＰＩ）和６周龄雏鸡静脉接种致病指数（ＩＶＰＩ）,结果ＭＤＴ为１０５小时、ＩＣＰＩ为１．３３、ＩＶＰＩ为１．０。试验结果表明本分离株为鸽新城疫病毒。     

单抗免疫过氧化物酶技术检测鸡传染性支气管炎病毒

以抗鸡传染性支气管病毒（ＩＢＶ）核衣壳蛋白（Ｎ）的单抗株６ＤＨ８作为一抗,以辣根过氧化物酶标记的羊抗鼠ＩｇＧ作为二抗,建立了检测石蜡切片中ＩＢＶ抗原的单抗免疫过氧化物酶技术（Ｍｃ－ＩＰ）,并对人工攻毒鸡及临床ＩＢＶ感染疑似鸡进行了检测。在ＩＢＶＭ４１株人工攻毒鸡,用该技术于１～１２ｄ从气管、２～７ｄ从肾脏可以检测到ＩＢＶ抗原,阳性染色集中于气管粘膜上皮细胞及肾小管上皮细胞胞浆;临床疑为ＩＢＶ感染的病鸡,以Ｍｃ－ＩＰ技术和单抗免疫荧光试验（Ｍｃ－ＩＦＡ）同时进行检测,结果阳性率分别为９０．３％及８３．９％。     

分泌抗鸡传染性支气管炎病毒单克隆抗体杂交瘤细胞株的建立

利用超速离心纯化的鸡传染性支气管炎病毒（ＩＢＶ）广东地方分离株Ｄ４１免疫ＢＡＬＢ／ｃ小鼠。取脾细胞与ＳＰ２／０骨髓瘤细胞进行融合,经筛选及克隆,成功地建立了６株可分泌抗ＩＢＶＤ４１株的单克隆抗体（ＭｃＡｂ）的杂交瘤细胞：Ｂ５、Ｂ８、Ｃ７、Ｄ８、Ｄ９、Ｇ１１。其中Ｂ８、Ｃ７、Ｇ１１的稳定性最好,腹水ＭｃＡｂ效价高达１０１０,无血清培养上清ＭｃＡｂ（浓缩３０倍）效价达４×１０４以上。６株杂交瘤细胞的特异性均很好。     

禽副粘病毒I型结构蛋白分析

鹅副粘病毒ＹＣ９７分离株,鸡源Ｖ９８分离株以及新城疫病毒（ＮＤＶ）Ｆ４８Ｅ８、ＬａＳＯｔａ和Ｃ３０毒株,鸽副粘病毒ＹＺＰＮＦ２分离株经鸡胚增殖纯化后,进行ＳＤＳ－ＰＡＧＥ电泳,结果显示ＹＧ９７和Ｙ９８２个毒株在５６ｋＤ处比另外４个毒株多出一个条带。用单抗２０１０株进行Ｗｅｓｔｅｒｎ ｂｌｏｔ转印,结果该单抗只对ＹＧ９７和Ｙ９８２个毒株的５６ｋＤ条带反应,证明这２株病毒的结构蛋白与经典的新城疫病毒有     

鉴别新城疫病毒弱毒株的抗多肽抗体制备

针对鸡新城疫病毒（ＮＤＶ）弱毒株Ｆ蛋白前体（Ｆ０）Ｆ２片段的特异结构,人工合成特异性多肽,将多肽与牛血清白蛋白（ＢＳＡ）化学偶联制和轩成全抗原后免疫小鼠,制备抗多肽血清。经ＥＬＩＳＡ检测,该抗体与ＭＤＶ弱毒株呈阳性反应,而与鸡痘病毒（ＦＰＶ）,鸡传染性腔上囊病病毒（ＩＢＤＶ）,ＭＤＶ强毒Ｆ４８Ｅ８株和四平析呈阴性反应,试验结果证明该抗体可以用于鉴别ＮＤＶ弱毒株。     

Genetic and antigenic diversity in avian infectious bronchitis virus isolates of the 1940s

In order to verify a commonly held assumption that only Massachusetts (Mass) serotype of infectious bronchitis virus (IBV) was prevalent in the United States between the 1930s (when IBV was first isolated) and the 1950s (when the use of commercial IBV vaccines began), we examined 40 IBV field isolates from the 1940s. Thirty-eight of those isolates were recognized as Mass serotype viruses based on their reactivity to Mass-specific monoclonal antibody (Mab) and neutralization by Mass-specific chicken serum. The remaining two isolates, N-M24 and N-M39, that did not react with Mass-specific Mab, resisted neutralization by Mass-specific chicken serum, and were neutralized only by homologous chicken antibody were identified as non-Mass IBV. When the first 900 nucleotides (nt) from the 5'-end of the spike (S1) glycoprotein gene and their deduced amino acid (aa) sequences were compared, the two non-Mass isolates differed from each other by 24% and 28%, respectively. In a similar comparison, the non-Mass viruses N-M24 and N-M39 differed from M28, a Mass-type isolate from the 1940s, by 21% and 22% (nt) and 28% and 27% (aa), respectively. These data indicate that antigenic and genetic diversity among IBV isolates existed even in the 1940s. Interestingly, when the N-terminal region of the S1 of M28 was compared to that of M41, a prototype Mass virus that has undergone countless number of in vivo and in vitro host passages, the two viruses differed by only 2% (nt) and 4% (aa). This finding suggests that frequent genetic changes are not inherent in all IBV genomes.     

Transmissibility of infectious bronchitis virus H120 vaccine strain among broilers under experimental conditions

The aim of this study was to quantify transmission of infectious bronchitis virus (IBV) H120 vaccine strain among broilers, and to assess whether birds that have been exposed to vaccine strain-shedding birds were protected against clinical signs after infection with a virulent strain of the same serotype. A transmission experiment and a replicate were carried out, each with six groups of commercial broilers. At day of hatch (n = 30) or at 15 days of age (n = 20), half of each group was inoculated with either IBV H120 vaccine (H120 group), virulent IBV M41 (M41 group), or were mock-infected, thereby contact-exposing the other half of each group. Nasal discharge was recorded, and antibody response and virus shedding were measured. To measure clinical protection, four weeks after inoculation all birds, in all groups, were challenged with IBV M41. The reproduction ratio (R; the average number of contact infections caused by one infectious bird) was determined to quantify virus transmission. All contact-exposed birds, except for one in an H120 group, became infected with either IBV H120 or IBV M41. Almost all birds contact-infected with IBV H120 or IBV M41 were subsequently protected against clinical signs after challenge with IBV M41. The lower limits of the 95% confidence interval (CI) of the R of IBV H120 vaccine, and of IBV M41, were significantly <1. For both IBV H120 and IBV M41, the 95% CI was [2.1-infinity] following inoculation at day of hatch and [1.8-infinity] after inoculation at 15 days of age. This finding demonstrates that IBV H120 vaccine is able to spread extensively among broilers. This implies that this vaccine strain might be able to become endemically present in the poultry population. It also implies that, even if not all birds received vaccine during spray application, due to the ability of the vaccine to spread in the flock, they will most likely be protected against clinical signs after a subsequent field virus infection.     

鸡传染性支气管炎病毒CQ/01/2004株的分离与鉴定

2004年从重庆某肉用鸡场疑似鸡传染性支气管炎的病鸡中采集病料，按常规处理后接种9～10日龄鸡胚，通过鸡胚连续传代培养3代，并对该分离毒株的鸡胚致病性、血凝性和NDV的干扰特性进行检测．同时进行了动物回归试验。结果表明，该分离株具有IBV感染特征，可使鸡胚胚体出血、蜷缩、矮化；该分离毒株无直接血凝性，对NDV有明显的干扰作用；动物回归试验中有75％的感染鸡在10d内发病或死亡。剖检病死鸡可见肾苍白、肿胀，肾小管内充塞大量尿酸盐，支气管有出血点、有大量粘液。采用反转录．聚合酶链式反应（RT-PCR）技术对CQ／01／2004的纤突蛋白S1基因进行扩增、克隆和序列测定，结果表明该基因具有IBVSl基因的共有分子特征，将测序结果提交GenBank进行同源性检索，发现分离株CQ／01／2004和J株S1的同源性最高，核苷酸同源性为94％，氨基酸同源性为89．4％，与M41的核苷酸同源性为80．6％，氨基酸同源性为78．0％。试验结果表明，分离的病毒株CQ／01／2004为鸡传染性支气管炎病毒。     

Production and characterization of monoclonal antibodies to (poly100)S1 protein of avian infectious bronchitis virus

Fragments within S1 genes ((poly100)S1) of infectious bronchitis virus (IBV) strains ZJ971, M41 and SC021202 (SC) were subcloned into a prokaryotic expression vector and expressed in Escherichia coli. Monoclonal antibodies (mAbs) against the recombinant (poly100)S1 proteins were produced, characterized and used to analyse epitopes on the S1 subunit of IBV. Nine mAbs raising from the three (poly100)S1 proteins recognized five different epitopes of the S1 subunit, designated as S1-A, B, C, D and E. Epitopes S1-C and S1-D are common for the three IBV strains, while S1-A and S1-B exist on ZJ971 and M41 strains, and S1-E was a strain-specific epitope for SC strain. Immunocytochemistry indicated that all the mAbs to the (poly100)S1 proteins can react with the homologous S1 glycoprotein expressed in Vero cells. Moreover neutralization test demonstrated that only mAbs 6E2, 4F9 and 6G4 had neutralization activity for the homologous IBV. These mAbs to (poly100)S1 protein were potential candidates for detecting and distinguishing IBV strains, and also used to examine antigenic variation of the S1 protein.     

鸡传染支气管炎病毒H52、H120和M41株的PCR鉴别方法研究

根据GenBank发表的序列,对鸡传染性支气管炎病毒（IBV）H52、H120和M41株全基因组序列进行比较,设计并合成了两对特异性引物,其中一对引物能以M41株为模板,特异性扩增出1791bp的目的片断,从而特异鉴定IBVM41株;另一对引物能以H52株和H120株为模板,特异性扩增1700bp的目的片断,再用限制性内切酶NaeI对PCR产物进行酶切,H120株能够被切成1000bp和700bp两个片段,而H52株的PCR产物不存在该酶切位点,从而能区分H52株和H120株。本研究建立的PCR方法和技术具有快速、简单、特异性强等优点,可用于IBVH52、H120和M41株活疫苗的分子鉴别。     

抗鸡传染性支气管炎病毒多克隆抗体的制备及其生物活性的鉴定

本试验将商品化疫苗株H120鸡传染性支气管炎病毒(IBV)直接免疫大白兔进行抗IBV全病毒粒子多克隆抗体的制备,通过琼扩试验及酶联免疫吸附试验检测其抗体效价,同时利用免疫组织化学、免疫荧光及Western blotting技术检测了所制备多抗血清的生物活性。试验结果表明,制备得到的多抗血清具有很高的抗体效价,同时能够与不同形式的IBV相关抗原发生特异性结合。     

鸡传染性支气管炎病毒GX-YL5株S蛋白在昆虫杆状病毒表达系统的表达及其免疫原性

为了对鸡传染性支气管炎病毒(avian infectious bronchitis virus,IBV)广西优势血清型代表株GX-YL5的S蛋白进行真核表达并研究其免疫原性,设计GX-YL5毒株S基因特异引物,扩增出目的片段后,构建重组表达载体pFastBacTM/HBM-TOPO-S,转化DH10Bac细胞获得重组杆...     

Maternal antibody to infectious bronchitis virus: its role in protection against infection and development of active immunity to vaccine

Chicks hatched with high levels of maternal antibody had excellent protection (>95%) against infectious bronchitis virus (IBV) challenge at 1 day of age, but not at 7 days (<30%). This protection significantly (P<0.05) correlated with levels of local respiratory antibody and not with serum antibody.A high percentage of both maternal antibody-positive (Mab+) and maternal antibody-negative (Mab-) chicks failed to produce IBV antibody when vaccinated at 1 day of age by the intraocular route. In addition, Mab+ chickens had a weaker virus-neutralizing antibody response to a second IBV vaccination compared to Mab- birds (P<0.05). Mab+ chicks experienced a more rapid decline (P<0.01) in maternal antibody after 1-day-of-age vaccination compared to their unvaccinated counterparts.A monoclonal antibody-based blocking ELISA that measured antibody levels specific to S1 glycoprotein of IBV correlated well with virus-neutralizing antibody titers.     

鸡传染性支气管炎病毒沈阳分离株生物学特性及其免疫原S1基因的克隆与序列分析

本研究对分离自沈阳地区的一株传染性支气管炎病毒（ＳＹ毒株）进行了生物学特性的研究,同时成功地对其免疫原Ｓ１基因进行了ＲＴ－ＰＣＲ扩增、克隆与序列分析。 通过电镜观察、动物回归试验、血凝特性研究等试验验证分离自沈阳地区的ＳＹ毒株确实为一株传染性支气管炎病毒。气管环组织培养交叉中和试验结果表明,分离株ＳＹ株不同于参考毒株澳大利亚Ｔ、Ｈ５２、Ｍ４１,且不同于国内其它流行株ＨＤ、ＨＢ、ＸＢ、ＤＢ等,是一个新的变异株。 利用ＩＢＶ Ｓ１基因特异性寡聚核苷酸引物,经ＲＴ－ＰＣＲ扩增ＳＹ毒株的Ｓ１基因,得到预期的约１．７Ｋｂ片段;并将扩增所得ｃＤＮＡ插入克隆质粒ｐＵＣ１９的ＥｃｏＲⅠ／ＢａｍＨⅠ位点,在大肠杆菌ＤＨ５ａ中实现目的基因的克隆。经限制性核酸内切酶分析及ＰＣＲ鉴定,证实为阳性重组质粒,利用末端双脱氧链终止法对其测序,得到Ｓ１基因全长１６４０ｂｐ,包括整个开放阅读框。通过序列分析软件ＤＮＡＳＩＳ、ＰＲＯＳＩＳ、ＭＥＧＡ等软件对Ｓ１基因核苷酸序列及推导的氨基酸序列进行分析,结果表明：分离株ＳＹ与７株参考株和国内流行株ＨＤ株相比,无论是核苷酸序列同源百分率还是氨基酸序列同源百分离都较低,均未达到８０％,这就提示我们ＳＹ毒