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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(ND1,226-4 916 bp)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pCI-ND1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1 200 bp大小的片段,经第一轮SOE-PCR得到ND1-1-2和ND1-3-4两个2 200 bp左右的片段,第二轮SOE-PCR得到4 900 bp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(226-4 916 bp),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 ND1-1,ND1-2,ND1-3,and ND1-4,and each fragment was amplified with PCR.SOE PCR was used to splice ND1-1 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 fragments 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 ND1-1,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 containing the full-length genome of the Newcastle disease virus.