【作者】 潘杰彦；

【导师】 陈溥言；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2001， 博士

【摘要】 实验选取了一株分离自有腺胃病变的较为特殊的传染性支气管炎病毒（IBVSD/97/02株）作为基本材料，对该毒株的生物学特性和分子生物学进行了研究。利用RT-PCR技术克隆了此毒株的所有结构蛋白（S1、S2、M、N）基因，并对这些基因的序列进行分析。内容包括：1.将病毒通过9-11日龄鸡胚盲传5-6代，用动物回归和免疫保护试验进行了毒株的基本特性的研究，用气管环组织培养对病毒进行理化特征测定、毒价测定与中和试验，初步证明目前流行的以腺胃肿大为主要特征的传染病的病原是冠状病毒，由于该病引起腺胃的特征性病理变化，所以将该病暂定名为腺胃型传染性支气管炎，并将该青岛分离株定为SD/97/02株。2 参考Genbank发表的IBV S1纤突蛋白基因序列，自行设计合成了一对引物，对传染性支气管炎病毒青岛腺胃分离株（SD/97/02）RNA进行RT-PCR扩增。产物经琼脂糖凝胶电泳分析，呈现一条1657 bp的条带，将其克隆入pMD18-T载体中，并进行序列测定，证实为S1基因。将此重组质粒命名为pMDQXS1。该序列已在Genbank上发表，号码为AF193423。序列分析表明，该每株S1基因G+C％含量较少，为37.0％，存在Hind Ⅲ，BamHⅠ，BgⅢ，SacⅠ和SalⅠ位点，无EcoRⅠ位点，与其他毒株的同源性在87.02％-94.21％之间，在第154-429nt处为高度变异区；将基因序列翻译成氨基酸后，S1蛋白由540个氨基酸组成，等电点为8.24，在蛋白质内部存在18个Cys，在S1与S2蛋白之间的剪切位点为HRRRR，这与大多数IBV毒株（RRF/SRR）不一样，有三个区域的氨基酸序列高度保守：即169-181aa，230-250aa，485-506aa。3.根据Genbank上发表的IBVS2基因序列，自行设计了两对引物，分别扩增SD/97/02株S2基因的上游1100bp部分和下游的900bp部分，两段序列拼接后包含了S2全基因的序列，将此两段分别克隆入pGEM T-easy载体，测序后将序列用分析软件DNASTAR和网上的BLAST软件进行分析，结果表明，S2基因比S1基因相对更保守，位于氨基酸第560-600位很可能是与囊膜锚着的部位。4.参考Genbank上发表的IBV核衣壳蛋白（N）基因序列，自行设计合成了一对引物，对青岛腺胃株病毒（QXIBV）RNA进行RT-PCR扩增，产物经琼脂糖凝胶电泳分析，呈现一条1248bp的条带，将其克隆入pGEM T-easy载体中，并用 Pst、Xbal、及Hind Ill对PCR产物进行酶切，然后进行序列测定，证实为N 基因。将此重组质粒命名为pGEMQXNP；序列分析表明，SD／97／02株的N基因 与Genbank上发表的序歹同源性在86－87％左右，在Z192nt，574－656nt， 68 1738nt处的变异最大，氨基酸同源性在89－gi％之间，在3－13aa，209－237aa 处变异较大；对N蛋白氨基酸特性分析表明，在N蛋白内部存在多个磷酸化位 点，且N蛋白具有强亲水性和强抗原性． 5．参考Genbank上的IBV序列，自行设计合成了三条引物，对传染性支气管炎青 岛腺胃分离株（SD／97／02）RNA进行 RT－PCR扩增，扩增含基质蛋白 ＊）及 sa、 sb蛋白的约1．65Kb的基因，对PCR产物进行克隆后测序。序列分析显示，M蛋 白基因与其它IBV相应的基因同源性在90．33％一92．75％之间，对氨基酸序列序 列比较，同源性在89．82％一94．25％之间；sa基因与其它IBV的基因同源性在 84．34％一87．37％之间，氨基酸同源性在8182％左右；sb基因与其它ISV的基因 同源性在引－92％之间，氨基酸同源性在93％左右。更多还原

【Abstract】 A new and specific strain of IBV causing proventriculitis was isolated from proventriculus and selected to study its biological characteristics and molecular biology.Ail structural protein genes(S 1.. S2~ M.~ N) were cloned by using the RT-PCR technique and then sequenced~. and analysed, All studies were listed below: l.A new kind of Disease occurred in the chick group in Qingdao area since Sep 1 996.The new virus isolate was propagated in the 9-11 -day-old chick embryo for 5-6 passages,and then several tests were done to identify the isolate, These tests include the test of returning virus to host; inirnuno-protectction test and using trachea ring culture to decide the titre of virus by viral neutralization test. All proved that the pathogen causing pathological lesion in proventriculus is a kind of avian coronavirus. Then this disease was temporally named as Infectious Bronchitis Proventiiculus Type, and this virus was designated as SD/97102(once called QXIBV) 2. A pair of primers were designed and synthesized according to the reported Si spike protein gene sequence of IBV. Then the viral RNA of QXIBV was amplified by reverse transcription-polymerase chain reaction(RT-PCR).The amplified product was analyzed by agarose gel electrophoresis, all appeared a fragment of 1657 bp as expected.The RT-PCR product was cloned into the pMDl 8-I vector ,and then the recombinant plasmid was sequenced.The result suggested that it was the SI spike gene 6 of IBV. The recombinant plasmid was designated pMDQXSI .The sequence has been published in Genbank,and the accession number is AF193423.Sequence analysis showed that the Si gene has low G+C contents at about 37.0%.There existed several RE cleavage sites such as Hind III, BamI-lI, B gill, Sac I and Sal I,but have no EcoR I site ,which is different from that of other strains. Its homology compared with other IBV strains was between 87.02% and 94.21%.In the site between 154 and 429 of this SI gene is a highly variation region. The SI protein is composed of 540 amino aicd residues after the gene was translated into amino acids.the p115 8.24.In the inner of the protein there exist 18 Cys residues.The sequence of the cleavage site between Si and S2 subunits is HRRRR,which is different from that of other IBV strains (RRFISRR). Three high conservative regions were located at 169-181,230-250.485-506 amino acids residues. 3. Two pairs of primers were designed to amp1if~?the upstream (1 lOObp)and downstream (900bp)sequences according to the reported IBV S2 gene sequence in Genbank. .The two sequences comprised the whole fragment of 52 subunit gene. All these two sequences were cloned into the pGEM I-easy vectors. The sequences were then analyzed using the software DNASTAR and BLAST. The result showed that the S2 gene was more conservative than Si gene .The amino acids located between 560-600 are probably associated with the envelope membrane. 4.A pair of primers were designed and synthesized according to the reported nucleocapsid protein(NP) gene sequence of IBV. Then the viral RNA of QXIBV was amplified by reverse transcription-polymerase chain reaction(RT-PCR).The amplified products were analyzed by agarose gel electrophoresis, all appeared a fragment of 1248 bp as expected.The RT-PCR products were cloned into the pGEM-T easy vector .The PCR products were digested with restriction endonuclease PstI,XbaI and HindIII,and the recombinant plasrnid was sequenced.The results suggested that it was the nucleoca更多还原