最早传入北京地区的SARS冠状病毒S基因序列分析和克隆

Sequencing and Cloning of S Gene of SARS Coronavirus First Found in Beijing Area

SARS冠状病毒的spike (S)蛋白对病毒的致病力至关重要 ,也是机体特异性体液和细胞免疫主要针对的靶分子。从北京地区最早发现的SARS患者咽拭子细胞培养上清中提取病毒RNA ,用反转录巢式聚合酶链式反应 (RT PCR)分 6个片段扩增出S基因全序列 ,用TA载体克隆后进行DNA序列分析 ,再通过重叠PCR将 6个片段连接成一条完整的S基因并克隆测序。DNA测序结果表明病毒S基因序列与报告的BJ0 1株SARS冠状病毒S基因序列完全一致 ,用重叠PCR将 6个S基因片段连接成了一条完整的S基因 ,插入到pGEM T载体后读序完全正确。上述结果表明最早传入北京地区的病毒与新近报告的BJ0 1株SARS冠状病毒在分子流行病学上具有同源特征 ,重叠PCR技术可以用于有效连接多个基因片段。S区全基因的克隆为进一步研究该基因的功能和DNA疫苗等研究提供了基础。

Spike (S) protein of SARS virus plays a key role in its virulence and is also the major target molecule for host humoral and cellular immune responses.The cultural supernatant of throat swab from a SARS patient first reported in Beijing area was used for viral RNA isolation.Nested RT-PCR was employed to amplify 6 overlapping fragments of S gene individually.The fragments were cloned into TA vector and then sequenced.Afterwards,overlapping PCR was performed to link the 6 individual fragments into an entire S gene.It was shown that the detected S gene sequence is identical in comparison with the one of BJ01 strain reported earlier.The linked entire S gene was proved with correct DNA sequence after inserted into pGEM-T vector.The results suggest the homologue between the analyzed virus and BJ01 strain in molecular epidemiology.Overlapping PCR technique proved to be practical to link several gene fragments.The entire S gene cloning is helpful for functional study of the gene.;