摘要
目的:利用CRISPR/Cas9技术构建ALF转录伸长因子4(AFF4)基因敲除的小鼠黑色素瘤细胞(B16-F10),自主制备特异的AFF4多克隆抗体。方法:设计一对分别靶向小鼠Aff4基因第3个外显子和第4个内含子的向导RNA(sgRNA)。将构建成功的重组质粒转染至B16-F10细胞中,并利用抗性筛选出单克隆细胞,进行基因型鉴定和AFF4表达水平检测。验证自主制备的AFF4多克隆抗体的特异性。结果:通过基因组DNA鉴定获得两株正确的单克隆细胞系,其Aff4基因mRNA水平(t=53.08,P<0.001)和AFF4蛋白表达水平(t=15.17,P<0.001)显著降低。免疫共沉淀实验和蛋白免疫印迹实验证明自主制备的抗人/鼠AFF4多克隆抗体特异性良好(t=264.7,P<0.0001)。结论:成功构建了Aff4基因敲除的B16-F10稳定细胞系,并且成功制备抗AFF4蛋白的多克隆抗体。
Objective:ALF transcription elongation factor 4(AFF4)gene knockout mouse melanoma cells(B16-F10)were constructed using CRISPR/Cas9 technology,and the specific AFF4 polyclonal antibody was prepared independently.Methods:A pair of guide RNA(sgRNA)targeting the third exon and the fourth intron of mouse Aff4 gene were designed.The successfully constructed recombinant plasmid was transfected into B16-F10 cells,and monoclonal cells were selected by resistance screening.The genotype was identified and the expression level of AFF4 was detected.The specificity of self-prepared AFF4 polyclonal antibody was verified.Results:Two correct monoclonal cell lines were obtained by genomic DNA identification,and their Aff4 mRNA level(t=53.08,P<0.001)and AFF4 protein expression level(t=15.17,P<0.001)were significantly decreased.Protein immunoprecipitation and Western blotting showed that the self-prepared anti-human/mouse AFF4 polyclonal antibody had good specificity(t=264.7,P<0.0001).Conclusion:Two Aff4 knockout B16-F10 cell lines are successfully established.The polyclonal antibody of AFF4 protein is successfully prepared.
作者
张佳慧
阎晗
胡德庆
ZHANG Jia-hui;YAN Han;HU De-qing(Department of Cell Biology,School of Basic Medical Sciences,Tianjin Medical University,Tianjin 300070,China)
出处
《天津医科大学学报》
2023年第4期372-378,共7页
Journal of Tianjin Medical University
基金
国家自然科学基金资助项目(31872825)。
