论文部分内容阅读
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 a prokaryotic expression plasmid encoding Cpf1 protein of Novicida subsp. Aureus, express it in E.coli and purify the recombinant Fn Cpf1 protein to prepare a polyclonal anti-Fn Cpf1 antibody. Methods: Fn Cpf1 gene was amplified by PCR and cloned into prokaryotic expression vector pET-32a (+). The recombinant plasmid was transformed into E. coli BL21 (DE3). IPTG induced the expression of target protein. Nickel metal- The purified Fn Cpf1 protein was purified and purified from New Zealand white rabbits to prepare Fn Cpf1 polyclonal antibody. The antiserum titer was detected by indirect ELISA and its specificity was identified by Western blot. Results: The 3 900 bp gene fragment was amplified and cloned into the expression vector pET-32a (+). After double digestion and sequencing, it was confirmed that the pET-32a (+) - Fn Cpf1 expression vector was constructed successfully, The molecular weight of 160 kD was analyzed by SDS-PAGE. The protein was purified by affinity chromatography with nickel ion metal chelate beads to obtain Fn Cpf1 protein with a purity of 95%. The polyclonal antibody was prepared and the rabbit anti-Fn Cpf1 Antiserum ELISA titer of 1: 512 000, Western blot results show that the prepared antibody can be better and Fn Cpf1 protein specific binding. CONCLUSION: Fn Cpf1 recombinant protein was successfully expressed in prokaryotic system and the purified antibody was used to prepare rabbit anti-Fn Cpf1 antibody. The titer and specificity of Fn Cpf1 antibody were good, which laid the experimental foundation for further exploring the biological characteristics of Cpf1 protein.