克里米亚-刚果出血热病毒特征核酸序列克隆载体的构建

Construction of cloning vectors containing necleotide sequence of Crimean-Congo hemorrhagic fever virus

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℃30 min,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。

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