马立克氏病病毒meq基因缺失株细菌人工染色体序列自我敲除的分子鉴定

doi:10.16431/j.cnki.1671-7236.2015.02.007

摘要/Abstract

摘要： 本试验旨在对缺失meq基因的马立克氏病病毒(MDV)感染性克隆基因组中细菌人工染色体(BAC)序列的自我敲除进行分子鉴定。将含有表达cre重组酶真核表达盒的SC9-1重组质粒通过转染鸡胚成纤维细胞(CEF)拯救SC9-1重组病毒。将SC9-1重组病毒在CEF细胞上进行连续传代培养,利用病毒表达的cre重组酶在传代过程中逐步敲除病毒的BAC序列。分别提取1~10代不同代次SC9-1重组病毒的DNA作为模板,利用BAC序列特异性引物对病毒基因组中的BAC进行PCR检测。利用针对BAC序列gpt基因的特异性检测引物,以MDV保守基因pp38作为内参,对不同代次病毒基因组中BAC序列进行荧光定量PCR的检测。利用PCR扩增SC9-1病毒敲除BAC序列后基因组中残留loxp位点两端序列,通过测序分析进一步验证BAC序列的敲除。利用BAC序列特异性引物对不同代次病毒基因组中BAC序列的检测结果显示,1~5代病毒DNA均能扩增出600 bp的特异性目的条带,证实病毒的基因组中含有BAC序列。6~10代病毒DNA均不能扩增出特异性目的条带,证实病毒基因组中没有BAC序列。荧光定量PCR结果显示,病毒基因组中的BAC序列随着病毒的传代逐渐减少,直至第6代已经完全检测不到BAC序列。在病毒BAC序列敲除的过程中,对每代病毒BAC序列敲除后残留loxp位点序列进行PCR测序鉴定,结果显示BAC序列敲除后基因组中仅留下一个loxp位点,序列同源性均在99.7%以上。结果表明,利用cre/loxp系统将病毒在CEF细胞上连续6代传代培养可将MDV meq基因缺失株SC9-1的BAC序列完全敲除掉,且同源重组酶敲除BAC序列具有高度一致性。

关键词: 马立克氏病; meq基因; 细菌人工染色体; 敲除; 鉴定

Abstract: The test was aimed to study the molecular identification of self-excision of bacterial artificial chromosome in meq-null Marek's disease virus (MDV) clone genome.SC9-1 recombinant plasmid containing cre recombinase expression cassette was transfected into chickens embryo fibroblast (CEF) to rescue SC9-1 recombinant virus.SC9-1 recombinant virus was passaged on CEF cells continuously,using cre recombinase knocked out BAC sequence in process of virus passaged.Extracting 1 to 10 generations DNA of SC9-1 recombinant virus as template,PCR detected BAC sequence of viral genome by specific primers.Using specific primers for the BAC sequence of gpt gene,the MDV conserved gene pp38 as an internal standard,the content of BAC sequences in the viral genome of different passages were detected by fluorescence quantitative PCR.The residual sequence of both sides of loxp site were amplified by PCR,and future verifying the knockout of BAC sequence by sequencing.The results showed that,using the BAC sequence specific primers to detect BAC sequence of different generations in the viral genome, the first five generation of virus DNA could amplify 600 bp specific band meaning the viral genome contained the BAC sequence.The 6 to 10 generationes of virus DNA couldn't amplify 600 bp specific band meaning the viral genome didn't contain the BAC sequence.The result of fluorescence quantitative PCR showed that the content of BAC sequence in viral genome decreased gradually with the passage of the virus and couldn't detect BAC sequence completely until the sixth generation.In the process of the knockout of BAC sequence,PCR identifying the residual sequence of both sides of loxp site,the results showed only one loxp site left after the knockout of BAC sequence and the sequence homology was above 99.7%.The experiment confirmed that the cre/loxp system could delete the BAC sequence in MDV meq-null mutant SC9-1 completely by culturing virus on CEF cells for 6 generation of batches and the knockout of BAC sequence had high degree of consistency.

Key words: Marek's disease; meq gene; bacterial artificial chromosomes; knockout; identification

中图分类号:

S852.65

孙鹏, 苏帅, 李延鹏, 丁家波, 崔治中. 马立克氏病病毒meq基因缺失株细菌人工染色体序列自我敲除的分子鉴定[J]. , 2015, 42(2): 285-291.

SUN Peng, SU Shuai, LI Yan-peng, DING Jia-bo, CUI Zhi-zhong. Molecular Identification of Self-excision of Bacterial Artificial Chromosome Sequence in Meq-null Marek's Disease Virus[J]. , 2015, 42(2): 285-291.

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