Cas9蛋白的克隆表达、分离纯化及多克隆抗体制备
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摘要
目的:获得高纯度Cas9蛋白,制备Cas9蛋白特异性抗体。方法:以Cas9基因序列为模板,设计特异性引物,PCR扩增获得Cas9基因全长序列,利用无缝拼接技术构建pET28a-Cas9重组表达载体;转化E.coli BL21(DE3),经IPTG诱导表达Cas9蛋白,并利用His-Tag技术分离纯化Cas9蛋白;将纯化获得的Cas9蛋白免疫新西兰大白兔,制备Cas9蛋白多克隆抗体。结果:pET28a-Cas9重组表达载体转化的E.coli BL21(DE3)可表达Cas9蛋白,纯化的Cas9蛋白免疫家兔得到的多克隆抗体效价>1∶256 K。结论:本研究获得了高质量Cas9蛋白及多克隆抗体,为后续CRISPR-Cas9基因编辑的应用奠定了良好的基础。
Objective:To obtain Cas9 protein with high-purity and prepare Cas9 protein-specific polyclonal antibody.Methods:The Cas9 gene sequences were used as a template to design specific primers,and the full-length sequence were obtained by PCR.The recombinant expression vector pET28 a-Cas9 was constructed by seamless splicing technique,and then transformed into E.coli BL21(DE3).The Cas9 protein was expressed by IPTG induction and purified by His-Tag technology.The purified Cas9 protein was finally used to immunize New Zealand white rabbits and to prepare polyclonal antibody.Results:The Cas9 protein was successfully expressed in E.coli BL21(DE3) and purified by His-Tag column.The polyclonal antibody was also successfully prepared with a titer of more than 1∶256 K.Conclusion:This study obtained Cas9 protein with high quality and polyclonal antibody,which laid a good foundation for the subsequent application of CRISPR-Cas9 gene editing.
Objective:To obtain Cas9 protein with high-purity and prepare Cas9 protein-specific polyclonal antibody.Methods:The Cas9 gene sequences were used as a template to design specific primers,and the full-length sequence were obtained by PCR.The recombinant expression vector pET28 a-Cas9 was constructed by seamless splicing technique,and then transformed into E.coli BL21(DE3).The Cas9 protein was expressed by IPTG induction and purified by His-Tag technology.The purified Cas9 protein was finally used to immunize New Zealand white rabbits and to prepare polyclonal antibody.Results:The Cas9 protein was successfully expressed in E.coli BL21(DE3) and purified by His-Tag column.The polyclonal antibody was also successfully prepared with a titer of more than 1∶256 K.Conclusion:This study obtained Cas9 protein with high quality and polyclonal antibody,which laid a good foundation for the subsequent application of CRISPR-Cas9 gene editing.
引文
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[23]YAO L,CENGIC I,ANFELT J,et al. Multiple gene repression in cyanobacteria using CRISPRi[J]. Acs Synthetic Biology,2016,5(3):1-22.
[2]WANG Y,ZHANG Z T,SEO S O,et al. Bacterial genome editing with CRISPR-Cas9:deletion,integration,single nucleotide modification,and desirable"clean"mutant selection in Clostridium beijerinckii as an example[J]. ACS Synth Biol,2016,5(7):721-732.
[3]KOMOR A,BADRAN A,LIU D. CRISPR-Based technologies for the manipulation of eukaryotic genomes[J].Cell,2017,168(1-2):20-36.
[4]KAVANAGH J N,REDMOND K M,SCHETTINO G,et al. DNA double strand break repair:a radiation perspective[J]. Antioxidants&Redox Signaling,2013,18(18):2458-2472.
[5]NISHIMASU,HIROSHI,CONG,et al. Crystal Structure of Staphylococcus aureus Cas9[J]. Cell,2015,162(5):1113-1126.
[6]CHEN R,XU Q,LIU Y,et al. Generation of transgenefree maize male sterile lines using the CRISPR/Cas9 system[J]. Front Plant Sci,2018,9:1180.
[7]HIRANO H,GOOTENBERG J,HORII T,et al. Structure and engineering of francisella novicida,Cas9[J].Cell,2016,164(5):950-961.
[8] OH J H,PIJKEREN J P V. CRISPR–Cas9-assisted recombineering in lactobacillus reuteri[J]. Nucleic Acids Research,2014,42(17):131.
[9] WANG Y,ZHANG Z T,SEO S O,et al. Markerless chromosomal gene deletion in Clostridium beijerinckii using CRISPR/Cas9 system[J]. Journal of Biotechnology,2015,200:1-5.
[10]COBB R E,WANG Y,ZHAO H. High-efficiency multiplex genome editing of streptomyces species using an engineered CRISPR/Cas system[J]. Acs Synthetic Biology,2015,4(6):723-728.
[11]HUANG H,ZHENG G,JIANG W,et al. One-step highefficiency CRISPR/Cas9-mediated genome editing in streptomyces[J]. Acta Biochim Biophys Sin,2015,47(4):231-243.
[12]TONG Y,CHARUSANTI P,ZHANG L,et al. CRISPRCas9 based engineering of actinomycetal genomes[J].ACS Synth Biol,2015,4(9):1020-1029.
[13]ZENG H,WEN S,XU W,et al. Highly efficient editing of the actinorhodin polyketide chain length factor gene in streptomyces coelicolor M145 using CRISPR/Cas9-CodA(sm)combined system[J]. Appl Microbiol Biotechnol,2015,99(24):10575-10585.
[14]CHAR S N,NEELAKANDAN A K,NAHAMPUN H,et al. An agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize[J]. Plant Biotechnology Journal,2017,15(2):257-268.
[15]LI J F,NORVILLE J E,AACH J,et al. Multiplex and homologous recombination-mediated genome editing in arabidopsis and nicotiana benthamiana using guide RNA and Cas9[J]. Nature Biotechnology,2013,31(8):688-691.
[16]MEI Y,WANG Y,CHEN H,et al. Recent progress in CRISPR/Cas9 technology[J]. Journal of Genetics and Genomics,2016,43(2):63-75.
[17]SU T,LIU F,GU P,et al. A CRISPR-Cas9 assisted non-homologous end-joining strategy for one-step engineering of bacterial genome[J]. Scientific Reports,2016,6:1-11.
[18]JIANG Y,CHEN B,DUAN C,et al. Multigene editing in the escherichia coli genome via the CRISPR-Cas9 system[J]. Applied&Environmental Microbiology,2015,81(7):2506-2514.
[19]CHOI K R,SANG Y L. CRISPR technologies for bacterial systems:Current achievements and future directions[J]. Biotechnology Advances,2016,34(7):1180-1209.
[20]OH-HASHI K,FURUTA E,FUJIMURA K,et al. Application of a novel Hi Bi T peptide tag for monitoring ATF4 protein expression in neuro2a cells[J]. Biochemistry and Biophysics Reports,2017,12:40-45.
[21]HOWDEN S,MCCOLL B,GLASER A,et al. A Cas9variant for efficient generation of indel-free knockin or gene-corrected human pluripotent stem cells[J]. Stem Cell Reports,2016,7(3):508-517.
[22]LIN S,EWEN-CAMPEN B,Ni X,et al. In vivo transcriptional activation using CRISPR-Cas9 in drosophila[J]. Genetics,2015,201(2):433-442.
[23]YAO L,CENGIC I,ANFELT J,et al. Multiple gene repression in cyanobacteria using CRISPRi[J]. Acs Synthetic Biology,2016,5(3):1-22.