IBV青岛腺胃分离株(SD/97/02)S1蛋白基因的序列测定和分析

Sequencing and Sequence Analysis of S1 Spike Protein Gene from Avian Infectious Bronchitis Virus Proventriculus Strain Isolated in Qingdao(SD/97/02)

参考Genbank上发表的IBVS1纤突蛋白基因序列 ,设计了一对引物 ,对鸡传染性支气管炎病毒青岛腺胃分离株 (SD/ 97/ 0 2 )RNA进行RT PCR扩增。将PCR产物克隆入pMD18 T载体中进行序列测定和分析。序列分析表明 ,该毒株的S1基因的G +C %含量较少 ,为 37.0 % ,存在HindIII,BamHI,BgIII,SacI和SalI位点 ,无EcoRI位点 ,与其他毒株的同源性在 87.0 2 % 94 .2 1%之间 ,在第 15 4～ 4 2 9nt处为高度的变异区 ;将基因序列翻译成氨基酸后 ,假定的S1蛋白由 5 4 0个氨基酸组成 ,等电点 8.2 4 ,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 ;16 9～ 181aa,2 30～ 2 5 0aa ,4 85～ 5 0 6aa ;与其他毒株进行抗原性比较后发现 ,在该毒株的 32 0～ 32 6aa及 390～ 4 0 1aa处的抗原表位消失 ,而在 32 5～345aa、379～ 389aa处则出现了很强的抗原表位 ;第 4 38～ 4 4 4aa处 ,其他IBV毒株 (除ZJ971株外 )原来存在的强抗原位点在本毒株中消失 ;在 5 3～ 6

A pair of primers were designed and synthesized according to the reported S1 spike protein gene sequence of IBV.The viral RNA of SD/97/02 strain 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 pMD18 T vector,and then the recombinant plasmid was sequenced.The result suggested that it was the S1 spike gene of IBV.The recombinant plasmid was designated pMDQXS1.The sequence has been published in the Genbank,and the accession number is AF193423.Sequence analysis showed that the S1 gene has low G+C content at about 37.0%.There existed several RE cleavage sites such as Hin d III, Bam H I, Bgl III, Sac I and Sal I,but have no Eco R 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 S1 gene is a highly variation region.The S1 protein is composed of 540 amino aicd residues after the gene was translated into amino acids,the pI is 8.24.In the inner of the protein there have 18 Cys residues.The sequence of the cleavage site between S1 and S2 subunits is HRRRR,which is different from that other IBV strains(RRF/SRR).Three high conservative regions were located at 169 181,230 250,485 506 amino acids residues.Comparative analysis of the antigenicity of SD/97/02 with that of other strains showed epitope disappearance in 320 326aa and 390 401aa,two strong epitopes appeared in 325 345aa and 379 389aa;in 438 444aa,the epitope which existed in other strains of IBV(except ZJ971 strain)disappeared;and the antigenicity between the amino acids residues 53 65 became weak.