应用SOE PCR方法构建新城疫病毒基因起始片段的研究

发布时间：2018-05-18 03:34

本文选题：新城疫病毒 + SOE　；参考：《中国病原生物学杂志》2017年03期

【摘要】：目的以猪源新城疫病毒JL02/2000株为模板,构建新城疫病毒反向遗传研究所需的基因起始片段。方法将NDV起始片段(ND1,226-4 916bp)设计拆分为ND1-1、ND1-2、ND1-3、ND1-4的4个约1.2kb的小片段,分别PCR扩增各片段,后采用SOE PCR技术将ND1-1、ND1-2拼接成中间片段ND1-1-2,将ND1-3、ND1-4拼接为ND1-3-4,再经第二次SOE-PCR整合拼接ND1-1-2和ND1-3-4片段,完成NDV起始片段ND1的构建,并连接到pCI载体上,构建pCIND1质粒,最后经酶切PCI-ND1质粒并送测序鉴定目的片段是否构建正确并成功连接到pCI载体上。结果经PCR扩增得到ND1-1、ND1-2、ND1-3、ND1-4共4个分别约1 200bp大小的片段,经第一轮SOE-PCR得到ND1-1-2和ND1-3-4两个2 200bp左右的片段,第二轮SOE-PCR得到4 900bp左右的ND1片段。连接产物PCI-ND1质粒经酶切和测序鉴定片段大小和序列与预期相符。结论成功构建了新城疫病毒基因起始片段(226-4 916bp),为构建新城疫病毒全长基因奠定了基础。
[Abstract]:Objective to construct the gene initiation fragment of Newcastle disease virus (NDV) by using JL02/2000 strain of Newcastle disease virus (NDV) as template. Methods four 1.2kb fragments of ND1-1 + Nd1-2Nd1-2Nd1-3 + Nd1-4 were designed by NDV starting fragment nd1226-4,916bp. each fragment was amplified by PCR. Then ND1-1Nd1-2 was spliced into ND1-1-2, ND1-3ND1-4 was spliced into ND1-3-4, and then ND1-1-2 and ND1-3-4 fragments were spliced into Nd1-3ND1-4 by the second SOE-PCR integration, and the fragments of Nd1-1nd1-2 and ND1-3ND1-4 were spliced into ND1-1-2and ND1-3ND1-4 by SOE PCR technique. The ND1 fragment of NDV was constructed and ligated to the pCI vector to construct the pCIND1 plasmid. Finally, the PCI-ND1 plasmid was digested and sequenced to confirm whether the target fragment was constructed correctly and successfully connected to the pCI vector. Results four fragments about 1 200bp in size were obtained by PCR amplification. Two 200bp fragments of ND1-1-2 and ND1-3-4 were obtained by the first round SOE-PCR, and the ND1 fragments of 4 900bp were obtained from the second round of SOE-PCR. The fragment size and sequence of the ligated PCI-ND1 plasmid were confirmed by restriction endonuclease digestion and sequencing. Conclusion the gene initiation fragment of Newcastle disease virus (NDV) was successfully constructed, which laid a foundation for the construction of the full-length gene of Newcastle disease virus (NDV).
【作者单位】：吉林大学动物医学学院;军事医学科学院军事兽医研究所;长春生物制品研究所有限责任公司;吉林农业大学动物科学技术学院;
【基金】：国家自然科学基金项目(No.31272573) 吉林省自然科学基金项目(No.20130101093JC) 吉林省科技发展计划项目(No.20140623019TC,20160623024TC)
【分类号】：S852.65

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