新型基因组编辑蛋白FnCpf1的表达及其功能的初步探讨
发布时间：2019-03-22 19:09
【摘要】：目的构建土拉弗朗西丝菌novicida亚种Cpf1蛋白编码基因的原核表达质粒,在大肠杆菌中诱导表达、纯化重组Fn Cpf1蛋白后,将纯化蛋白免疫新西兰大白兔制备兔抗Fn Cpf1多克隆抗体,间接ELISA法和Western blot分析制备抗体的免疫反应性及特异性。并对FnCpf1功能做初步的探讨。方法1.以土拉弗朗西丝菌novicida亚种基因组为模板,利用PCR技术扩增Fn Cpf1基因,将其克隆到原核表达载体p ET-32a(+)中,并用双酶切和测序验证。2.将构建成功的pET-32a(+)-FnCpf1重组质粒转化大肠杆菌BL21(DE3),IPTG诱导目的蛋白的表达,通过镍离子金属螯合磁珠亲和纯化FnCpf1蛋白。3.将纯化蛋白浓缩液与弗氏佐剂充分混合,免疫新西兰大白兔制备FnCpf1多克隆抗体,并用间接ELISA法检测兔抗血清效价、Western blot鉴定其特异性。4.构建一个插入失活的mcherry荧光报告载体,将其转化至表达fncpf1蛋白的大肠杆菌感受态中,用肉眼直接观察是否出现红色菌落以判断是否进行了切割、重组。结果1.扩增得到3900bp目的基因片段,克隆到表达载体pet-32a(+)后,通过双酶切鉴定大小与预期一致,测序结果与genbank一致,证实pet-32a(+)-fncpf1表达载体构建成功。2.可诱导表达相对分子质量约为160kd的目的蛋白,经sds-page分析其为可溶性,通过镍离子金属螯合磁珠亲和纯化得到fncpf1纯化蛋白,imagelab的体积工具分析结果表明纯度为95%以上,bca法测得超滤浓缩后的纯化蛋白浓度为1.4mg/ml。3.用纯化蛋白免疫新西兰大白兔3次后,制备多克隆抗体并检测兔抗fncpf1抗血清elisa效价达1:512000,westernblot结果显示制备的抗体可较好地与fncpf1蛋白特异性结合。4.将插入失活的mcherry荧光报告载体转化至表达fncpf1蛋白的大肠杆菌感受态中,平板上可见红色菌落长出,而转化至大肠杆菌dh5α的则未见红色菌落。结论1.成功构建了fncpf1重组表达载体,并在原核系统中诱导、表达和纯化fncpf1重组蛋白。2.用纯化后的蛋白制备出兔抗fncpf1抗体,效价及特异性均良好,为进一步探讨cpf1蛋白的生物学特性打下实验基础。3.在表达FnCpf1的大肠杆菌中,利用CRISPR-Cpf1系统对插入失活的mCherry红色荧光报告基因进行靶向切割并重组,证明了FnCpf1蛋白的核酸酶活性。
[Abstract]:Objective to construct a prokaryotic expression plasmid encoding Cpf1 gene of novicida subspecies in E. coli, and to express the recombinant Fn Cpf1 protein in E. coli, then immunize New Zealand rabbits with purified Fn Cpf1 protein to prepare rabbit anti-Fn Cpf1 polyclonal antibody. Indirect ELISA and Western blot were used to analyze the immuno-reactivity and specificity of the antibody. The function of FnCpf1 is also discussed. Method 1. Using novicida subspecies genome as template, Fn Cpf1 gene was amplified by PCR and cloned into prokaryotic expression vector p ET-32a (), and verified by double enzyme digestion and sequencing. The recombinant plasmid pET-32a ()-FnCpf1 was successfully constructed and transformed into E. coli BL21 (DE3), IPTG-induced target protein expression). FnCpf1 protein was purified by Ni ~ (2 +) metal chelating magnetic beads affinity. 3. FnCpf1 polyclonal antibody was prepared by immunizing New Zealand rabbits with purified protein concentrate and Freund's adjuvant. The specificity of the purified protein concentrate was determined by, Western blot of rabbit antiserum titer. An inactivated mcherry fluorescent report vector was constructed and transformed into the competent state of E. coli expressing fncpf1 protein. The presence of red colony was observed directly by naked eye to determine whether it had been cut and recombined. Outcome 1. The target gene fragment of 3900bp was amplified and cloned into the expression vector pet-32a (). The size was confirmed to be consistent with the expected size by double enzyme digestion, and the sequencing results were consistent with that of genbank. It was confirmed that the expression vector pet-32a ()-fncpf1 was constructed successfully. The fncpf1 purified protein was purified by Ni ~ (2 +) metal chelating magnetic beads. The volume tool analysis of imagelab showed that the purity of imagelab was more than 95%, and the protein was soluble by sds-page. The relative molecular weight of 160kd could be induced to express the protein, which was soluble by sds-page and purified by Ni ~ (2 +) metal chelating magnetic beads. The concentration of purified protein after ultrafiltration was 1.4 mg / ml 路3. 3. The concentration of purified protein determined by bca method was 1.4 mg / ml. After immunizing New Zealand rabbits with purified protein for three times, polyclonal antibody was prepared and the titer of elisa in rabbit anti-fncpf1 serum reached 1? 512000. Western blot results showed that the prepared antibody could bind to fncpf1 protein specifically. 4. When the inserted inactivated mcherry fluorescent report vector was transformed into the competent state of E. coli expressing fncpf1 protein, red colony was observed on the plate, but no red colony was found when transformed into E. coli dh5 伪. Conclusion 1. The recombinant expression vector of fncpf1 was successfully constructed and induced, expressed and purified in prokaryotic system. 2. The recombinant protein of fncpf1 was expressed and purified. Rabbit anti-fncpf1 antibody was prepared from purified protein with good titer and specificity, which laid the experimental foundation for further study on the biological characteristics of cpf1 protein. 3. In E. coli expressing FnCpf1, CRISPR-Cpf1 system was used to target cleavage and recombination of the inactivated mCherry red fluorescence reporter gene, which proved the nuclease activity of FnCpf1 protein.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378
本文编号：2445839
[Abstract]:Objective to construct a prokaryotic expression plasmid encoding Cpf1 gene of novicida subspecies in E. coli, and to express the recombinant Fn Cpf1 protein in E. coli, then immunize New Zealand rabbits with purified Fn Cpf1 protein to prepare rabbit anti-Fn Cpf1 polyclonal antibody. Indirect ELISA and Western blot were used to analyze the immuno-reactivity and specificity of the antibody. The function of FnCpf1 is also discussed. Method 1. Using novicida subspecies genome as template, Fn Cpf1 gene was amplified by PCR and cloned into prokaryotic expression vector p ET-32a (), and verified by double enzyme digestion and sequencing. The recombinant plasmid pET-32a ()-FnCpf1 was successfully constructed and transformed into E. coli BL21 (DE3), IPTG-induced target protein expression). FnCpf1 protein was purified by Ni ~ (2 +) metal chelating magnetic beads affinity. 3. FnCpf1 polyclonal antibody was prepared by immunizing New Zealand rabbits with purified protein concentrate and Freund's adjuvant. The specificity of the purified protein concentrate was determined by, Western blot of rabbit antiserum titer. An inactivated mcherry fluorescent report vector was constructed and transformed into the competent state of E. coli expressing fncpf1 protein. The presence of red colony was observed directly by naked eye to determine whether it had been cut and recombined. Outcome 1. The target gene fragment of 3900bp was amplified and cloned into the expression vector pet-32a (). The size was confirmed to be consistent with the expected size by double enzyme digestion, and the sequencing results were consistent with that of genbank. It was confirmed that the expression vector pet-32a ()-fncpf1 was constructed successfully. The fncpf1 purified protein was purified by Ni ~ (2 +) metal chelating magnetic beads. The volume tool analysis of imagelab showed that the purity of imagelab was more than 95%, and the protein was soluble by sds-page. The relative molecular weight of 160kd could be induced to express the protein, which was soluble by sds-page and purified by Ni ~ (2 +) metal chelating magnetic beads. The concentration of purified protein after ultrafiltration was 1.4 mg / ml 路3. 3. The concentration of purified protein determined by bca method was 1.4 mg / ml. After immunizing New Zealand rabbits with purified protein for three times, polyclonal antibody was prepared and the titer of elisa in rabbit anti-fncpf1 serum reached 1? 512000. Western blot results showed that the prepared antibody could bind to fncpf1 protein specifically. 4. When the inserted inactivated mcherry fluorescent report vector was transformed into the competent state of E. coli expressing fncpf1 protein, red colony was observed on the plate, but no red colony was found when transformed into E. coli dh5 伪. Conclusion 1. The recombinant expression vector of fncpf1 was successfully constructed and induced, expressed and purified in prokaryotic system. 2. The recombinant protein of fncpf1 was expressed and purified. Rabbit anti-fncpf1 antibody was prepared from purified protein with good titer and specificity, which laid the experimental foundation for further study on the biological characteristics of cpf1 protein. 3. In E. coli expressing FnCpf1, CRISPR-Cpf1 system was used to target cleavage and recombination of the inactivated mCherry red fluorescence reporter gene, which proved the nuclease activity of FnCpf1 protein.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378
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1 孙菱;新型基因组编辑蛋白FnCpf1的表达及其功能的初步探讨[D];重庆医科大学;2017年
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