SARS冠状病毒N基因的扩增与克隆

南方医科大学学报 ›› 2004, Vol. 24 ›› Issue (01): 39-41.

SARS冠状病毒N基因的扩增与克隆

肖维威¹, 马文丽¹, 张宝¹, 王艳¹, 毛向明¹, 彭翼飞¹, 宋艳斌¹, 吴清华¹, 郑文岭²

1. 第一军医大学分子生物学研究所, 广东, 广州, 510515;
2. 广州军区广州总医院分子肿瘤学研究所, 广东, 广州, 510010

出版日期:2004-01-20 发布日期:2004-01-20
基金资助:
收稿日期:2003-7-1。
作者简介:肖维威(1974- ),女,1998年毕业于第一军医大学,现为第一军医大学在读博士研究生,讲师,电话:020-61648114-89097
通讯作者:马文丽,电话:020-61648210

Amplification and cloning of the N gene of SARS-associated coronavirus

XIAO Wei-wei¹, MA Wen-li¹, ZHANG Bao¹, WANG Yan¹, MAO Xiang-ming¹, PENG Yi-fei¹, SONG Yan-bin¹, WU Qing-hua¹, ZHENG Wen-ling²

1. 第一军医大学分子生物学研究所, 广东, 广州, 510515;
2. 广州军区广州总医院分子肿瘤学研究所, 广东, 广州, 510010

Online:2004-01-20 Published:2004-01-20

摘要/Abstract

摘要： 目的 RT-PCR扩增、克隆SARS冠状病毒N蛋白基因。方法根据GenBank数据库中TOR₂株的全基因组序列,利用Primer Premier 5.0软件设计引物RT-PCR巢式扩增SARS冠状病毒的N基因,PCR产物克隆后进行测序鉴定。结果序列分析表明,pMD18-T载体中已成功重组了N基因。结论 N蛋白基因的扩增、克隆成功,为N蛋白的表达、N蛋白结构与功能的研究奠定了基础。

Abstract: Objective To amplify and clone the N g en e of severe acute respiratory syndrome-associated coronavirus.Method Using prim er Premier 5.0 software, two pairs of nested PCR primers were designed to amplif y the N gene. After purification, the amplified products were cloned into pMD18 -T vectors, and the positive clones with the inserted fragments were identified by sequence analysis.Results The amplified products was about 1 375 bp in leng th, and sequence analysis demonstrated that the N gene fragments had been succe ssfully inserted into pMD18-T vectors.Conclusion The successful amplification a nd cloning of N gene facilitates further investigation of the expression of the N protein and study of its structure and functions.

中图分类号:

Q786

肖维威¹, 马文丽¹, 张宝¹, 王艳¹, 毛向明¹, 彭翼飞¹, 宋艳斌¹, 吴清华¹, 郑文岭². SARS冠状病毒N基因的扩增与克隆[J]. 南方医科大学学报, 2004, 24(01): 39-41.

XIAO Wei-wei¹, MA Wen-li¹, ZHANG Bao¹, WANG Yan¹, MAO Xiang-ming¹, PENG Yi-fei¹, SONG Yan-bin¹, WU Qing-hua¹, ZHENG Wen-ling². Amplification and cloning of the N gene of SARS-associated coronavirus[J]. Journal of Southern Medical University, 2004, 24(01): 39-41.

参考文献

[1] Peiris J,Lai S,Poon L,et al. Coronavirus as a possible cause of severe acute respiratory syndrome[J]. Lancet,2003,361 (9366):1319-25.
[2] Ksiazek TG,Erdman D,Goldsmith CS,et al. A novel coronavirus associated with severe acute respiratory syndrome[J]. N Engl J Med,2003,348(20):1953-66.
[3] Rota PA,Oberste MS,Monroe SS,et al. Characterization of a novel coronavirus associated with severe acute respiratory syndrome[J].Science,2003,300(5624):1394-9.
[4] Nelson GW,Stohlman SA,Tahara SM.High affinity interaction between nucleocapsid protein andleader/intergenic sequence of mouse hepatitis virus RNA[J]. J Gen Virol,2000,81(Pt1):181-8.
[5] Chen H,Wurm T,Britton P,et al.Interaction of the coronavirus nucleoprotein with nucleolar antigens andthe host cell[J]. J Virol,2002,76(10):5233-50.
[6] 王艳,马文丽,宋艳彬,等.反转录巢式PCR方法检测SARS冠状病毒[J].第一军医大学学报,2003,23(5):421-3.Wang Y,Ma WL,Song YB,et al.Gene sequence analysis of SARS-associated coronavirus by nested RT-PCR[J]. J First Mil Med Univ/DI Yi Jun Yi Da Xue Xue Bao,2003,23(5):421-3.
[7] 车小燕,丘立文,潘玉先,等.SARS冠状病毒N蛋白单克隆抗体的快速、高效制备的方法研究[J].第一军医大学学报,2003,23(7):640-2.Cbe XY,Qiu LW,Pan YX,et al. Rapid and efficient preparation of monoclonal antibodies against SARS-associated coronavirus nucleocapsid protein by immunizing mice[J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao,2003,23(7):640-2.
[8] 车小燕,郝卫,丘立文,等.SARS病人SARS冠状病毒核壳抗原抗体的变化规律[J].第一军医大学学报,2003,23(7):637-9.Che XY,Hao W,Qiu LW,et al. Antibody response of patients with severe acute respiratory syndrome (SARS) to nucleocapsid antigen of SARS-associated coronavirus[J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao,2003,23(7):637-9.

[1]	张雷；陈建平；张莉；王涛；刘明杰；田玉； . mompS-linker-flaA融合基因原核表达载体的构建及诱导表达[J]. 南方医科大学学报, 2006, 26(12): 1701-1705.
[2]	李海玉；郭爱华；刘志锋；刘瑜；刘靖华；邓鹏；李志杰；刘亚伟；姜勇；. 细胞穿透肽核靶向运输蛋白表达载体的构建及其蛋白转导功能的研究[J]. 南方医科大学学报, 2006, 26(10): 1394-.
[3]	任淑婷；于琳华；徐长福；高广道；. 诱导性鼠Smad7真核表达载体的构建与鉴定[J]. 南方医科大学学报, 2006, 26(09): 1313-1315.
[4]	李黎波¹, 冯茹², 周淑芸². 重组绿色荧光蛋白腺相关病毒载体转染293细胞和CD34⁺细胞[J]. 南方医科大学学报, 2005, 25(01): 26-29.
[5]	姚红兵, 吴爱国, 黄宗海. Survivin在结直肠癌中的表达及其临床意义[J]. 南方医科大学学报, 2004, 24(12): 1412-1415.
[6]	刘忠英¹, 罗深秋¹, 邓凡¹, 赵永忠¹, 李西平². 新生大鼠磷脂酶C-γ1mRNA的表达变化[J]. 南方医科大学学报, 2004, 24(09): 987-990.
[7]	赵海金¹, 蔡绍曦¹, 邹飞², 佟万成¹, 万为人². 应用Super SMART cDNA合成技术扩增哮喘嗜酸性粒细胞总RNA[J]. 南方医科大学学报, 2004, 24(09): 1037-1039.
[8]	张嘉杰¹, 吴少瑜¹, 徐伟¹, 侯金林², 王战会², 姜世勃³, 吴曙光¹. SARS冠状病毒S蛋白S1区多肽与SARS病人血清的免疫反应[J]. 南方医科大学学报, 2004, 24(07): 789-790,797.
[9]	盛小刚¹, 冯建章¹, 吴书林¹, 靳立军¹, 余细勇², 张斌¹. 骨髓间充质干细胞的肌源性诱导分化及转染VEGF基因的表达[J]. 南方医科大学学报, 2004, 24(03): 290-294.