猪繁殖与呼吸综合征病毒CH-1a株非结构基因的分子克隆及其基因特征的研究

Molecular Cloning and Sequence Analysis of the NSP Coding Region of Porcine Reproductive and Respiratory Syndrome Virus CH-1a Strain

对猪繁殖与呼吸综合征病毒的非结构基因 (ORF1)和非编码区分别进行了克隆和测序 ,并对其基因特征作了分析。结果表明 ,CH_1a株ORF1全长 11882nt,其中ORF1a长 75 12nt、ORF1b长 4374nt。它们与VR2 332株的核苷酸同源性分别为 89%、92 % ;与LV株的核苷酸同源性分别为 5 4%、6 3.3%。ORF1编码两个聚合蛋白 ,其中ORF1a编码的聚合蛋白经裂解后产生 6个成熟的非结构蛋白 (Nsp1α、Nsp1β、Nsp2_5 ) ,以Nsp2的变异性最为显著 ,它与LV株的氨基酸同源性为 41%。ORF1b编码的聚合蛋白经裂解后 ,产生 4个成熟的非结构蛋白 (RdRpCP2_4) ,其中RdRp为病毒的复制酶 ,CP4表现出较大的变异性 ,与LV株的氨基酸同源性为 48%。在ORF1a_ORF1b的衔接区有保守的庚核苷酸滑动序列和拟节结构 ,它们是核糖体移码翻译所必需的结构。在基因组末端存在非编码区 ,其中 5’NCR长 190nt,比LV株 5’NCR短 31nt,其核苷酸同源性为 6 1%。 3’NCR长 15 1nt,比LV株 3’NCR长 37nt,保守性稍高 ,其与VR2 332株和LV株的核苷酸同源性分别为 95 .4%和 78.2 %。在 3’NCR末端有一个poly(A)尾巴 ,长 2 0nt。在Poly(A)尾上游有保守的八核苷酸序列 ,是复制酶识别并结合的区域。

In the study,the non-structural protein coding regions,including ORF1,5'Non-Coding Region(NCR) and 3'NCR of porcine reproductive and respiratory syndrome virus (PRRSV) strain CH-1a were sequenced and analyzed.The results showed that 5'NCR of CH-1a is 190nt in length.It shared 94% nucleotide identity with strain VR2332,a representative of North American genotype.It was shorter than that of strain LV,a representative of European genotype,and shared only 61% nucleotide identity.ORF1 was also determined.The results showed that ORF1 is 11882nt in length.ORF1a and ORF1b are 7512nt and 4373nt in length,respectively.ORF1a and ORF1b of CH-1a shared 89% and 92% nucleotide identities with VR2332,respectively.However,the sequence was distinct from LV,ORF1a and ORF1b of CH-1a exhibited only 54% and 63.3% nucleotide identities with LV. ORF1a of CH-1a codes a polyprotein.Six putative Nsps (Nsp1α,Nsp1β and Nsp2-5) are predicted.Nsp2 is the most divergent among different PRRSV strains.Nsp2 of CH-1a shared 81% and 41% amino acid identities with VR2332 and LV,respectively.ORF1b codes another polyprotein.Four small putative proteins (RdRp,CP2-4) are predicted.Compared with ORF1a,ORF1b is more conserved.But the carboxyl-terminal of CP4 is distinct,sharing only 48% amino acid identity with LV.The ORF1a-1b frame shift region of PRRSV is highly conserved.A hepta-nucleotide'slippery'sequence located just upstream of the stop codon of ORF1a,and a pseudo-knot structure downstream of the slippery sequence were believed to be essential for the expression of ORF1b via a mechanism of ribosomal frame shifting.The 3'NCR of CH-1a is 151 nucleotides in length,and was 37 nucleotides longer than that of LV.It shared 78.2% and 95.4% nucleotide identities with LV and with VR2332,respectively.There is a poly(A) tail at the 3'-eod.A motif,consisting of eight nucleotides immediately upstream of the poly(A),is identical to those of LV and VR-2332.The results showed that the genetic distance between CH-1a and LV was further than that between CH-1a and VR2332,indicating that CH-1a and VR2332 can be classified to the same genotype.