摘要
应用Gibson Assembly连接法精确快速构建狂犬病病毒SRV9株重组感染性cDNA克隆,为狂犬病病毒感染性cDNA克隆的构建提供新的方法。应用Gibson Assembly连接法在同一反应体系内,将多个片段和经限制性内切酶线性化的载体,按设计的顺序进行连接,实现多个片段的一步组装。设计带有20bp^30bp重叠序列的PCR引物,扩增得到带有重叠序列的狂犬病病毒5个结构蛋白基因和增强型荧光蛋白基因。扩增的片段和线性化的载体经胶回收纯化后,与Gibson连接液混合后,50℃反应60min,连接产物转化Stbl3感受态细胞,提取重组质粒,经PCR、酶切和测序鉴定,缺失了病毒伪基因Ψ区和糖蛋白基因的跨膜区并且将增强型荧光蛋白基因插入糖蛋白基因终止密码子前,构建了全长17 600bp的重组质粒,完成了重组全长感染性cDNA克隆的构建。
The recombinant full-length infectious cDNA clone of SRV9 rabies virus was constructed by Gibson Assembly,which provided a new method for the construction of rabies virus infectious cDNA clone.A few fragments and the enzyme linearized carriers were connected by Assembly Gibson connection method in the same reaction system according to the design of connection order for the realization of one step assembly of a number of fragments. PCR primers were designed with 20-30bp overlap sequence, and five structural protein genes and fluorescent protein gene with overlapping sequences were amplified.The amplified fragments and linearized vectors after purification by gel extraction were mixed and connected to the Gibson liquid,reacting at 50℃ for 60 min,the products were transformed into Stbll3 competent cells.The recombinant plasmid was identified by PCR,enzyme digestion and sequencing.After the deletion of the glycoprotein gene transmembrane region and virus pseudo gene ψ region inserting enhanced green fluorescent protein gene into ahead the glycoprotein gene stop codon, the 17 600 bp recombinant plasmid and the recombinant infectious full length cDNA clone were constructed successfully and quickly.
出处
《动物医学进展》
北大核心
2017年第11期11-17,共7页
Progress In Veterinary Medicine
基金
国家自然科学基金项目(31360623)
新疆生产建设兵团应用基础研究项目(2016AG008)
