应用SOE PCR方法构建新城疫病毒基因起始片段的研究  

Research on using SOE PCR to construct initial fragments of genes of the Newcastle disease virus

作　　者：南福龙 陈兴[2,3] 张赫[2] 解长占 崔卓栋 张萍[2,4] 张金勇[2,4] 庄忻宇 鲁会军[2] 金宁一[1,2] 

机构地区：[1]吉林大学动物医学学院,吉林长春130062 [2]军事医学科学院军事兽医研究所,吉林长春130122 [3]长春生物制品研究所有限责任公司,吉林长春130012 [4]吉林农业大学动物科学技术学院,吉林长春130118

出　　处：《中国病原生物学杂志》2017年第3期193-196,共4页Journal of Pathogen Biology

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)

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。Objective To synthesize initial fragments of genes of the Newcastle disease virus （JL02/2000） in order to create a construct containing the full-length genome of the virus. Methods Initial fragments of the ND1 gene （226--4 916 bp） were divided into four smaller fragments designated NDI-1, ND1-2, ND1-3, and ND1-4, and each fragment was amplified with PCR. SOE PCR was used to splice NDI-1 and ND1-2 to an intermediate ND1 1-2. ND1-3 and ND1-4 were spliced to ND1-3-4. Two intermediates, ND1-1,2 and ND1-3,4, were assembled with SOE PCR to synthesize initial frag- ments of the ND1 gene, and the ND1 fragment was ligated to a pCI vector to construct the plasmid pCI-ND1. The target fragment was identified with sequencing as well as restriction enzyme digestion. Results NDI-1, ND1-2, ND1-3, and ND1 4 were amplified with PCR, yielding a product of about 1 200 bp. SOE PCR was used to synthesize two intermediates, ND1-1-2 and ND1-3-4, yielding a fragment of 2 200 bp each. An ND1 fragment of about 4 900 bp was synthesized using a second round of SOE PCR. The pCI-ND1 plasmid was identified with sequencing and restriction enzyme digestion, and the results of enzyme digestion and genetic sequencing were consistent with expectations. Conclusion Initial fragments of a gene were successfully spliced using SOE PCR. This work has laid the foundation for creating a construct con- taining the full-length genome of the Newcastle disease virus.

关 键 词：新城疫病毒 SOE PCR 基因克隆 拼接 

分 类 号：R37[医药卫生—病原生物学]