利用Gibson Assembly法构建狂犬病SRV9ψ区缺失株感染性cDNA

发布时间：2018-01-22 21:54

本文关键词： Gibson Assembly连接法狂犬病病毒感染性cDNA 连接　出处：《基因组学与应用生物学》2017年06期 　论文类型：期刊论文

【摘要】：本研究为高效构建狂犬病SRV9ψ区缺失株感染性cDNA克隆,在细胞内进行重组狂犬病病毒的拯救提供帮助。按照Isothermal in vitro recombination system or"Gibson Assembly"连接方法设计引物,分别扩增得到去除狂犬病SRV9标准疫苗株的伪基因区(ψ区)的首尾均存在互补序列的5个结构蛋白基因的目的片段。利用T5核酸外切酶、DNA聚合酶及T4连接酶的协同作用,两步即可获得狂犬病SRV9ψ区缺失株的感染性cDNA。本研究利用Gibson Assembly连接法成功高效构建了狂犬病SRV9ψ区缺失株的感染性cDNA。此方法避免了传统的酶切连接方法中繁琐的实验步骤,实现了多个基因片段间的无缝连接,大大提高了载体构建的效率,为进一步研究狂犬病病毒的基因功能提供高效的方法。同时也为Gibson Assembly法应用于其它载体的构建提供借鉴。
[Abstract]:The aim of this study was to construct an infectious cDNA clone of rabies SRV9 蠄 deletion strain. The rescue of recombinant rabies virus in the cell was carried out according to Isothermal in vitro recombination system or. The primer was designed by Gibson Assembly. Five structural protein genes with complementary sequences were amplified from the pseudo gene region (蠄 region) of rabies SRV9 standard vaccine strain. T5 nucleic acid exonuclease was used. The synergism of DNA polymerase and T4 ligase. Infectious cDNA of rabies SRV9 蠄 deletion strain could be obtained in two steps. Gibson was used in this study. The infectious cDNA of rabies SRV9 蠄 deletion strain was successfully constructed by Assembly ligation method. This method avoids the tedious experimental steps in the traditional enzyme ligation method. The seamless connection between multiple gene fragments is realized, and the efficiency of vector construction is greatly improved. It provides an efficient method for further study on the gene function of rabies virus, and also provides a reference for the application of Gibson Assembly method in the construction of other vectors.
【作者单位】：新疆农业大学动物医学学院;新疆农垦科学院畜牧兽医研究所;
【基金】：国家自然科学基金“犬瘟热病毒N基因-犬细小病毒VP2基因构建重组狂犬病病毒的研究”(31360623)资助
【分类号】：R373
【正文快照】： 狂犬病病毒(rabies virus,RV)为单股负链RNAprotein,N)、磷酸化蛋白(phosphoprotein,P)、基质蛋白病毒,基因组共编码五个结构蛋白:核蛋白(nucleo-(matrix protein gene,M)、糖蛋白(glycoprotein gene,G) 和多聚酶蛋白(RNA dependent RNA polymerase,L)因的PCR产物条带大小分

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