李云峰，蔡鹏，臧宇婷，石楠，葛藤，薛芳，宋锋林，吴刚,于畅

    辽宁国际旅行卫生保健中心，辽宁 大连　116001

    摘要：目的 构建含有克里米亚-刚果出血热病毒特征序列的克隆载体。方法设计RT-PCR引物及反应体系，从克里米亚-刚果出血热病毒RNA中扩增获得目的序列，进行分离纯化和回收，将纯化的扩增片段与pMD18-T载体进行连接，进行酶切鉴定与测序鉴定。结果确定检测克里米亚-刚果出血热病毒一步法RT-PCR的反应条件及体系，引物终浓度为400 nmol/L，反应条件为：50℃ 30min, 94℃ 4 min, 94℃ 30s, 57℃ 30 s, 72℃ 30 s,35cycles.成功将CCHF的特征序列片段克隆，pMD18-T载体经DNA测序鉴定正确。结论构建的pMD18-CCHF质粒，可用于克里米亚-刚果出血热病毒RT-PCR实验的阳性对照。

   关键词：克里米亚-刚果出血热；克里米亚-刚果出血热病毒；逆转录聚合酶链反应；核酸序列；克隆载体；检测

    中图分类号：R512.8　文献标识码：B

    Construction of cloning vectors containingnecleotide sequence

    of Crimean-Congo hemorrhagic fever virus

    LI Yun-feng, CAI Peng, ZANG Yu-ting, SHI Nan,GE Teng, XUE Fang, SONG Feng-lin, WU Gang，YU Chang

    Liaoning International Travel HealthcareCenter, Dalian, Liaoning 116001, China

    Abstract:   Objective  To construct a coloning vectors containing necleotide sequence ofCrimean-Congo hemorrhagic fever virus.  Methods  The primers and reaction system of one step RT-PCR to explore thebest detection condition were designed, and the products werecloned into pMD18-T vector. Recombinant plasmid named pMD18-CCHFwas identified by enzyme digestion and sequencing analysis. Results   The best detection condition of RT-PCR forCrimean-Congo hemorrhagic fever virus was as following: primersfinal concentration 400 nmol/L, amplifying sequence: 50℃ 30 min,94℃ 4 min, 94℃ 30s, 57℃ 30 s, 72℃ 30 s, 35cycles. The coloningvector containing the necleotide sequence of Crimean-Congohemorrhagic fever virus was constructed successfully. Conclusion   This recombinant vector pMD18-CCHF can beused as positive control in the RT-PCR test for Crimean-Congohemorrhagic fever virus.

    Key words:   Crimean-Congohemorrhagic fever virus;  RT-PCR; Nacleotide sequence; Cloningvectors

    《中国国境卫生检疫杂志》2012年6月刊