摘要
应用Crispr/Cas9基因编辑技术敲除MDCK细胞中的Annexin A2基因,并验证其是否为ε毒素在MDCK细胞表面的受体。根据Crispr/Cas9靶点设计原则设计靶点,构建PALentiCRISPR v2重组质粒。借助Crispr/Cas9技术,将构建好的质粒转染至MDCK细胞中,用嘌呤霉素筛选敲除Annexin A2蛋白的MDCK细胞株,并通过测序和Western blot验证敲除效果。通过细胞毒性试验验证MDCK细胞膜表面的Annexin A2蛋白是否为ε毒素的受体蛋白。测序结果和Western blot结果表明,通过Crispr/Cas9技术成功敲除了MDCK细胞的Annexin A2基因,但细胞毒性试验结果表明,敲除Annexin A2基因并不能减弱ε毒素对MDCK细胞的毒性。结果表明,Annexin A2蛋白不是ε毒素在MDCK细胞表面的受体,研究结果为今后MDCK细胞其他蛋白的敲除提供了试验方法,构建的Annexin A2基因敲除的MDCK细胞也为Annexin A2蛋白引起的其他疾病的研究奠定了基础。
To knock out Annexin A2 gene in MDCK cells using clustered regularly interspaced short palindromic repeats(Crispr)/Cas9 gene-editing system and to verify whether the ε-toxin is the receptor in MDCK cell line,firstly,the target sequence was designed according to the target-designing rules of Crispr/Cas9 and cloned into the PALentiCRISPR v2 recombinant plasmid.The plasmid was transfected into MDCK cells.The Annexin A2-knocking out cell strains were screened through the stress of puromycin,and the knockout effect was detected by sequencing and Western blotting.Finally,whether the Annexin A2 is the receptor of ε-toxin in MDCK cells is tested through the cell viability with recombinant toxin.The results of sequencing and Western blotting indicated that the Annexin A2-knocking out MDCK cell line was successfully constructed using Crispr/Cas9 system.However,the test of cell viability showed that the knockout of Annexin A2 cannot decrease the potent of ε-toxin on MDCK cells.Annexin A2 is not the receptor of ε-toxin in MDCK cells,nevertheless,this paper provided an efficient protocol to knock out a target gene in MDCK cells.In addition,Annexin A2 knockout MDCK cell line could be used in other studies associated with Annexin A2.
作者
黄静
高洁
夏苏苏
金志颖
康琳
高姗
杨浩
辛文文
赵宝华
王景林
HUANG Jing;GAO Jie;XIA Su-su;JIN Zhi-ying;KANG Lin;GAO Shan;YANG Hao;XIN Wen-wen;ZHAO Bao-hua;WANG Jing-lin(State Key Laboratory of Pathogen and Biosecurity,Institute of Microbiology and Epidemiology,AMMS,Beijing,100071,China;Life Science Institute of Hebei Normal University,Shijiazhuang,Hebei,050024,China)
出处
《动物医学进展》
北大核心
2019年第5期13-17,共5页
Progress In Veterinary Medicine
基金
国家自然科学基金项目(31500108)
