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鸡传染性支气管炎病毒核蛋白基因的克隆及在杆状病毒系统中的表达 总被引：5，自引：1，他引：4
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Study of protection by recombinant fowl poxvirus expressing C-terminal nucleocapsid protein of infectious bronchitis virus against challenge. 总被引：10，自引：0，他引：10
A stable recombinant fowl poxvirus (rFPV) expressing the C-terminal region (119 amino acids) of the nucleocapsid (N) protein of an infectious bronchitis virus (IBV) strain Ch3 was constructed by inserting the coding sequence within the thymidine kinase gene of fowl poxvirus (FPV) by homologous recombination. The N protein was expressed under control of the vaccinia virus promoter P7.5 in chicken embryo fibroblast cell cultures as seen in immunofluorescence assay and in rFPV-inoculated specific-pathogen-free (SPF) chickens by detecting antibodies with enzyme-linked immunosorbent assay (ELISA). A homologous IBV strain (Ch3) and two heterologous IBV strains (Ch5 and H4) were used to inoculate SPF chickens in a challenge to examine the protective efficacy of the rFPV. When the chickens were challenged with IBV Ch3 or Ch5, the control birds had respiratory signs of infections bronchitis, whereas all the vaccinated birds were clinically normal although low levels of the IBV infection were detected by a differential ELISA. In contrast, in the chickens challenged with IBV H4, all control birds and vaccinated birds suffered from the highly lethal IBV H4 infection. Our results suggest that the C-terminal 119 amino acid of the nucleocapsid expressed by FPV is a host-protective antigen and may induce cross-protective immunity against illness among some IBV strains. 相似文献
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Using the whole infectious bronchitis virus (IBV) for detecting the antibody against IBV by enzyme-linked immunosorbent assay (ELISA) is a routine work in poultry industry. To prepare virus is time consuming and tedious. Furthermore, the whole viral antigen detects all antibodies against the viral structural proteins, including spike (S), nucleocapsid, matrix, and other proteins. Among those, S protein is related to neutralization. Thus, to develop and express protein fragment from S gene and to use the protein as a coating antigen for antibody detection against IBV are the purposes of this experiment. A partial S gene fragment (n.t. 1143-1665) was cloned into pRSET vectors and transformed into competent Escherichia coli (E. coli) BL21 (DE3). A 27.5 kDa fusion protein (S-fg, containing S1-F and partial S2-G antigenic sites) was successfully expressed, affinity-purified and detected specifically with chicken anti-IBV serum by Western blot. The expressed S-fg protein was used as a coating antigen for developing an ELISA (S-fg ELISA) for serum antibody detection in anti-IBV antisera from different IBV serotypes and in field sera. The results show that the S-fg fusion protein is highly cross-reactive among different IBV serotypes, and the S-fg ELISA is found to be a convenient, economical, and efficient method for antibody detection against IBV. 相似文献
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鸡传染性支气管炎病毒分离株核衣壳基因克隆、序列分析和真核表达 总被引：2，自引：0，他引：2
根据GenBank公开序列自行设计一对引物，采用RT—PCR扩增出鸡传染性支气管炎病毒（Infectious bronchitis virus，IBV）W和C9001分离株完整的核衣壳（N）基因，并将其克隆至pMD18-T载体进行核苷酸序列测定和分析。结果表明，扩增的2个IBV分离株核衣壳基因片段长度均为1230bp，编码409个氨基酸，彼此间核苷酸和氨基酸同源性分别为88．0％和89．5％，与GenBank中有代表性的参考毒株相应基因核苷酸和氨基酸序列比较显示，w株核苷酸序列与GenBank中的广东分离株（AY646283）同源性最高，为94．1％，氨基酸序列同源性为94．6％；与国内部分毒株核苷酸序列同源性在86．1％～88．0％之间，氨基酸序列同源性在88．0％～90．7％之间；C9001株与国内部分毒株核苷酸序列同源性在86．4％～99．8％之间，氨基酸序列同源性在88．0％～99．8％之间。从核衣壳基因编码的氨基酸序列的系统进化树可见，W株与C9001株处于不同的进化分枝，亲缘关系较远。同时将核衣壳基因构建于真核表达质粒pVAX1中，用脂质体法将重组质粒转染入COS-7细胞中，间接免疫荧光检测出核衣壳蛋白的体外表达。研究结果为进一步研究IBV核衣壳蛋白的结构与功能以及基因工程疫苗的研制奠定了基础。 相似文献
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Molecular epidemiology of infectious bronchitis virus isolates from China and Southeast Asia 总被引：14，自引：0，他引：14
In order to trace the origin and evolution of avian infectious bronchitis virus (IBV) isolates in China and Southeast Asia, genomic sequencing was used for molecular characterization of 24 IBV isolates and two reference strains in comparison with the published sequences. The 5' region of the S1 genes, containing hypervariable regions I and II, and 3' region of the nucleocapsid genes, containing cytotoxic T lymphocyte epitopes, were used to construct phylogenetic trees for analysis. The results showed that the 24 isolates could be divided into three distinct groups, that is, American, Asian, and European. Some isolates formed a distinct Asian phylogenetic group, suggesting that IBV has existed for some time in Asia. Our results also showed that in vivo recombination of IBV may have occurred at a rather high frequency, contributing to the diversity of these IBV isolates. Importantly, recombination events have probably occurred between vaccine strains and field strains in the natural condition. 相似文献
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我国鸡传染性支气管炎病毒地方性流行毒株S1基因的RT-PCR扩增及其克隆与鉴定 总被引：2，自引：0，他引：2
采用ＲＴ－ＰＣＲ方法,以ＩＢＶＳ１全基因特异性引物分别从我国华东（ＨＤ）、华北（ＨＢ）、华中（ＨＺ）、华南（ＨＮ）、西北（ＸＢ）及东北（ＤＢ）等地的ＩＢＶ流行株基因组中扩增出预期的１．７ｋｂ左右的ＤＮＡ片段。ＰＣＲ产物的ＨａｅＩＩ酶切分析及其与英国ＩＢＶＳ１全基因核酸探针的分子杂交证实所获６个ＩＢＶ流行株的ＰＣＲ产物为ＩＢＶＳ１基因。将此６个毒株的Ｓ１基因ＰＣＲ产物分别进行５’和３’端的ＢａｍＨＩ和ＨｉｎｄＩＩ酶切识别位点的分子修饰之后插入到克隆质粒ｐＵＣ１８的ＢａｍＨＩ／ＨｉｎｄＩＩ位点,在大肠杆菌中实现了目的基因的分子克隆。Ｓ１基因的ＲＦＬＰ分析表明我国ＩＢＶ已有分子水平的变异。 相似文献
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Infectious bronchitis virus (IBV) was detected by indirect immunofluorescent assay with a monoclonal antibody (MAb-IFA). The monoclonal antibody was specific for the nucleocapsid protein of IBV strain M41. The MAb-IFA clearly detected IBV with high specificity in infected chicken kidney cells. The assay furthermore detected IBV in tracheal smears and sliced tracheas from experimentally infected chickens. The positive reaction was found to be longer than that in the virus recovery test. These results indicate that MAb-IFA is a useful method for the detection of IBV from chickens suspected to have infectious bronchitis. 相似文献
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单抗免疫过氧化物酶技术检测鸡传染性支气管炎病毒 总被引：11，自引：3，他引：8
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％。 相似文献
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Isolation and identification of four infectious bronchitis virus strains in China and analyses of their S1 glycoprotein gene 总被引：4，自引：0，他引：4
Between 2003 and 2005, four strains of infectious bronchitis virus (IBV) were isolated from the vaccinated chicken flocks in China. The results from chicken embryo cross-neutralization assays showed that all the four isolates were relative to strain A2 of IBV, which was isolated in 1996 in Beijing and related to strain 4/91. The S1 gene of the spike protein was amplified and sequenced. The nucleotide and amino acid sequence of the S1 gene had a similar degree of identity (88.98-99.28%) among the four Chinese IBV isolates. The identity of the S1 protein gene between the four Chinese IBV isolates and 14 strains of other IBVs varied from 70.06 to 81.59%. Phylogenetic analysis suggested that there are at least four groups of IBVs circulating in China and the disease outbreaks might have been caused by infection of multiple strains of IBV. 相似文献
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根据国外已发表的鸡传染性支气管炎病毒（ Ｉ Ｂ Ｖ）核蛋白基因序列,设计并合成 １ 对引物。应用这对引物,以 Ｒ Ｔ Ｐ Ｃ Ｒ 特异性扩增出华南分离株（ Ｈ Ｎ 株）的核蛋白基因,基因产物大小为 １５ ｋｂ , 与设计相符。对 Ｈ Ｎ 株部分核蛋白基因进行序列测定,并与标准毒株 Ｈ５２、 Ｍ ４１、 Ｂｅａｕ 和 Ｇｒａｙ 的核蛋白基因进行同源性比较,结果表明, Ｈ Ｎ 株与 Ｈ５２、 Ｍ ４１、 Ｂｅａｕ 和 Ｇｒａｙ 株的核酸同源性分别为 ８５％ 、８４％ ８４％ 和 ８６％ 。由此可以看出, Ｈ Ｎ 株与标准毒株在核蛋白基因上存在一定的差异。 相似文献
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本试验根据已公布的IBV株S1基因序列及pPIC9K表达载体序列，去掉由18个氨基酸构成的信号肽后，设计一对IBV S1基因表达片段的PCR引物，利用RT—PCR扩增得到了IBV四川分离株的S1基因片段，将该片段克隆到PMD18-T载体上，通过对所得到的重组质粒进行酶切分析、菌落PCR鉴定，证明得到了含有目的基因片段的阳性重组质粒，测序分析片段长为1566bp，已经成功切除了由18个氨基酸构成的信号肽序列。将该基因亚克隆到毕赤酵母分泌型表达载体pPIC9K的SnaBⅠ和NotⅠ酶切位点，并通过菌落PCR、双酶切鉴定了该重组质粒的正确性，IBV S1基因毕赤酵母表达载体的构建为进一步利用毕赤酵母表达IBV S1蛋白提供了基础材料，并对表达产物的免疫原性和禽传染性支气管炎基因亚单位疫苗及特异性诊断抗原的研究打下了基础。 相似文献
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Fifty-six isolates of avian infectious bronchitis virus (IBV) were obtained from different field outbreaks in China in 2010, and they were genotyped by comparison with 19 reference strains in the present study. The results showed that LX4-type isolates are still the predominant IBVs circulating in chicken flocks in China, and these isolates could be grouped further into two clusters. Viruses in each cluster had favored amino acid residues at different positions in the S1 subunit of the spike protein. In addition, a recombination event was observed to have occurred between LX4- and tl/CH/LDT3/03I-type strains, which contributed to the emergence of a new strain. The most important finding of the study is the isolation and identification of Taiwan II-type (TW II-type) strains of IBV in mainland China in recent years. The genome of TW II-type IBV strains isolated in mainland China has experienced mutations and deletions, as demonstrated by comparison of the entire genome sequence with those of IBV strains isolated in Taiwan. Pathogenicity testing and sequence analysis of the 3' terminal untranslated region revealed that TW II-type IBV strains isolated in mainland China have a close relationship with the embryo-passaged, attenuated TW2296/95. 相似文献
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对自1994—2009年从我国5省区免疫鸡群中分离到的37株传染性支气管炎病毒(IBV)的S蛋白基因序列进行分析,发现S1基因序列存在广泛的氨基酸替换、缺失和插入现象,大部分IBV分离株S1基因的推导氨基酸序列变异主要集中在60～63、73～74、97、128、282～299位等。S2基因较为保守,主要在裂解位点后的2～47、122～152位发生氨基酸的替换,可见IBV S基因的不断变异可能是造成本试验所调查的5省区免疫鸡群IB频发的重要原因。遗传进化分析发现本试验所调查地区近十多年来肾型毒株仍是主要流行株,没有或少有4/91型毒株流行。所调研地区鸡的腺胃炎持续广泛地发生,但是从临床腺胃病料中很少分离到IBV,可见IBV不太可能是引起腺胃炎的主要病原。 相似文献
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上海地区鸡传染性支气管炎病毒S2基因的检测及分析 总被引：1，自引：1，他引：0
参考GenBank发表的传染性支气管炎病毒（infectious bronchitis virus，IBV）S基因序列，设计合成了1对引物，模拟PCR表明，该引物能扩增现已发表的所有IBV的S2基因部分核酸序列，扩增片段493bp。建立的RT—PCR检洲体系检测灵敏度达10～50ELD50，检出限量为0．1ng。采集上海地区12个鸡场疑似IBV禽样品34份进行检洲，阳性为32份，序列分析表明均为肾变型IBV S2基因片段，S2基因同源性较高（≥99％），与其他地区肾变型IBV也有较高同源性，而与腺胃型和呼吸型相比较，序列差异较大。进化分析表明，尽管IBV S2片段变异率明显低于S1基因，但二者表现相同的进化趋势，S2基因亦可反映出IBV的分子变异及毒株间的亲缘关系。 相似文献
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1996-2008年从我国不同地区分离30株传染性支气管炎病毒（Infectious Bronchitis Viruses,IBV）野毒株的M基因,采用RT-PCR方法克隆测定所分离的野毒株和澳大利亚T株的M基因序列,利用生物信息学软件与GenBank中公布的部分国内外IBV毒株的M基因序列进行比较分析,研究我国IBV的分子流行学特点和分子遗传变异规律。结果发现所测毒株M基因具有4种不同长度的开放阅读框：669bp、672bp、678bp和681bp,分别编码222、223、225和226个氨基酸的多肽,这些长度的差异是由5′端的核苷酸插入或缺失造成的。30个IBV分离株间的同源性在89．5％～100％之间。以疫苗株H120氨基酸位置为参照,在被比较的73株IBVM蛋白中发现62个位点存在变异,其中以2～5、10～16、44～46、217～222等4个区域氨基酸取代率较高。系统进化分析显示,被比较的73个IBV毒株分为5个进化群,我国的IBV分属于其中的4个群,其中第一群和第四群与我国所使用的疫苗病毒株相距较远。同时发现部分近年的分离株与10多年前分离株具有很近遗传进化关系。从M基因看,在我国出现了多种基因型IBV共存的现象,分离株与疫苗株的遗传差异提示我们需要对疫苗的选用做出重新评估。 相似文献