含非洲马瘟病毒部分核酸序列的病毒样颗粒的研制

Preparation of the Virus-like Particles Containing the Partial Gene Fragment of AHSV

VP7蛋白是非洲马瘟病毒(AHSV)群特异性蛋白,其编码基因S7基因常被用作AHSV RT-PCR和实时荧光RT-PCR检测的靶标基因。本研究旨在构建耐RNase的内含AHSV部分核酸序列的病毒样颗粒。首先合成S7基因保守序列,然后克隆到含有噬菌体包膜蛋白基因的带组氨酸纯化标签的假病毒表达载体pNH-MS2his上,成功构建原核表达载体pNH-MS2his-VP7。将重组质粒pNH-MS2his-VP7转化BL21(DE3),并进行诱导表达及镍离子亲和层析纯化后,得到含AHSV部分RNA片段的病毒样颗粒。试验证实该病毒样颗粒均匀性和稳定性良好,可作为AHSV PCR检测的质控品和标准品使用。

This experiment was conducted to prepare the virus-like particles containing the partial gene fragment of African horse sickness virus(AHSV).Genome segment 7(S7)encoding protein VP7,the group-specific protein of AHSV,is a primary target for reverse transcription-PCR and real time reverse transcription-PCR detection of AHSV.The conservative sequence of S7 gene was synthesized and cloned into plasmid pNH-MS2 his,which contained the coat protein gene of E.coli bacteriophage MS2 and His tag,to construct the prokaryotic express vector pNH-MS2hisVP7.The recombinant plasmid pNH-MS2his-VP7 was transformed into BL21(DE3).Expression of the target protein was induced by IPTG for 5h.The virus-like particles containing the S7 RNA sequence of AHSV were simply obtained by induction and sequent Ni+chromatography purification.The virus-like particles were confirmed have good uniformity and stability.They have great prospect as the standard and quality control in the detection of AHSV.