新型基因组编辑蛋白FnCpf1的表达、鉴定及多克隆抗体的制备

Prokaryotic expression and the antiserum preparation of FnCpf1,a novel genome editing protein encoding gene

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,在大肠杆菌中表达、纯化重组Fn Cpf1蛋白后,制备兔抗Fn Cpf1多克隆抗体。方法:利用PCR技术扩增Fn Cpf1基因,将其克隆到原核表达载体pET-32a(+)中并转化大肠杆菌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得到纯度较高的Fn Cpf1蛋白,将纯化蛋白免疫新西兰大白兔制备Fn Cpf1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。结果:扩增得到3 900 bp的目的基因片段,克隆到表达载体pET-32a(+)后,通过双酶切及测序证实pET-32a(+)-Fn Cpf1表达载体构建成功,可诱导表达相对分子质量160 k D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纯度为95%以上的Fn Cpf1蛋白,制备多克隆抗体并检测兔抗Fn Cpf1抗血清ELISA效价达1:512 000,Western blot结果显示制备的抗体可较好地与Fn Cpf1蛋白特异性结合。结论:在原核系统中成功表达了Fn Cpf1重组蛋白,并用纯化后的蛋白制备出兔抗Fn Cpf1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。

Objective：To construct the prokaryotic expression vector of Cpf1 of Francisella tularensis subsp. Novicida,to express the recombinant protein in E.coli BL21 and purify it to prepare FnCpf1 polyclonal antibody. Methods：FnCpf1 gene was amplified by PCR and subcloned into a prokaryotic expression vector pET-32a（＋）. Then the pET-32a（＋）-FnCpf1 was transformed into E. coli BL21 and the recombinant FnCpf1 was induced with IPTG. The FnCpf1 protein was purified by the nickel ion chelating beads affinity purification and was used to immunize rabbits. The titer and specificity of the polyclonal antibody was respectively measured by Indirect enzyme-linked immunosorbent assay（ELISA）and Western blot. Results：Restriction and sequencing analysis proved that recombinant plasmid pET32a（＋）-FnCpf1 was constructed correctly. SDS-PAGE analysis showed that FnCpf1 with a relative molecular mass of 160 k D was expressed in a soluble form. The purity of the purified protein reached more than 95 percent. ELISA showed the titer of FnCpf1 polyclonal antibody was 1∶512 000 and Western blot test demonstrated that the polyclonal antibody could specifically bind with FnCpf1 recombinant protein. Conclusion：FnCpf1 recombinant protein is successfully expressed in the prokaryotic system and FnCpf1 polyclonal antibody is prepared with high titer and specificity,which would lay the foundation for further study on the biological characteristics of Cpf1.