传染性支气管炎病毒核衣壳蛋白基因变异及在大场杆菌中的表达(英文)

Variation of nucleocapsid protein gene of infectious bronchitis virus and its expression in Escherichia coli

核衣壳蛋白 (N)是传染性支气管炎病毒的主要结构蛋白之一。以前的研究结果显示 IBV- ZJ971毒株以引起腺胃肿大为特征 ,IBV- X和 N毒株以引起肾炎为特征。为了探讨不同组织嗜性传染性支气管炎病毒 N基因的变异 ,我们依据已发表的 IBV- Beaudette毒株 N基因的序列设计和合成引物 ,RT-PCR扩增 IBV- ZJ971、N、X和 H5 2的核衣壳蛋白基因 ,并测序、分析和在 E.coli中进行表达。结果显示 :IBV- ZJ971、N、X和 H5 2毒株的 N基因的 ORF由 12 30 bp组成 ,编码 4 0 9个氨基酸。与来源于 Gen Bank中的 IBV毒株比较 ,IBV- ZJ971、N、X和 H5 2与其它 IBV毒株的核苷酸同源性分别是 87.0 - 98.6 %、86 .6 - 99.7%、86 .3- 99.7%、87.2 % - 98.5 % ,氨基酸的同源性分别是 90 .0 - 97.8%、 90 .5 - 98.8%、 90 .0 -98.8%、 91- 98%。IBV- ZJ971毒株的 N蛋白不同于其他 IBV的独特变异是 111,117,14 2 ,171和 4 0 1位点上的氨基酸改变 ,IBV- N和 X毒株的 N蛋白不同与其它 IBV的独特变异是 4 6 ,4 8,189,190 ,2 2 0 ,2 2 3,2 36 ,2 37,2 4 0 ,2 86 ,2 99,30 1334,335 ,336 and35 1位点上的氨基酸改变。说明 IBV嗜腺胃毒株(ZJ971)和嗜肾毒株 (N和 X)的 N基因以散在的点突变为特征。将 IBV- ZJ971毒株的 N基因在 E.

Nucleocapsis (N) protein gene is one of the major structural protein genes of infectious bronchitis virus (IBV) Our previous studies have indicated that IBV strain ZJ971 causes proventriculus lesions while strains N and X result in chicken nephritis This research was undertaken to investigate their variance where two specific primers were designed and synthesized according to the published sequence of N protein gene of IBV strain Beaudette cDNAs of N gene were amplified by reverse transcription polymerase chain reaction (RTPCR) from viral RNAs of IBVZJ971, N, X and H52 strains, cloned into vector pBluescript SK (+) and sequenced Sequencing results showed that complete open reading frame (ORF) of N gene encode a protein of 409 amino acids that was 1230 bp in length from IBV strains ZJ971, N, X and H52 When the nucleotide and predicted amino acid sequence of N gene is compared with that of the known IBV strains in GenBank, the homology of nucleotides range from 870% to 986%, 866% to997%, 863% to 997% and 872% to 985% for ZJ971, N, X and H52, respectively Furthermore the similarities of predicted amino acids range from 900% to 978%, 905% to 988%, 900% to 988% and 91% to 98% respectively Based on the analysis of the predicted amino acid sequence, it was noted that IBV strain ZJ971 was different from the other 11 IBV strains in that mutation occured at sites 111,117, 142, 171 and 401 Remarkable variation of amino acids, distinct from 10 IBV strains shown, were observed at sites 46, 48, 189,190, 220, 223, 236, 237, 240, 286, 299, 301 334, 335, 336 and 351 cDNA of N gene for ZJ971 strain was subcloned into prokaryotic expression vector pBV220 Upon expression in Escherichia coli DH5α, a specific nonfusion protein of 45 kD was detected by westernblotting assays using monoclonal antibody against N protein of IBV These studies suggested that IBV strains ZJ971, N and X were three novel IBV strains of China origin 