The Study On The Expression And Characteristic Of FnCpf1,a Novel Genome Editing Protein

ObjectivesThis study was aimed to construct the prokaryotic expression vector of Cpf1 protein from Novicida,which was the subspecies of Francisella tularensis.After expressed in E.coli BL21 and purified,the recombinant protein Fn Cpf1 was immunized with the New Zealand white rabbits to prepare FnCpf1 polyclonal antibody.And then the immunogenicity and specificity of this antibody were detected by indirect enzyme-linked immunosorbent(ELISA)and Western blot and do a preliminary study of the function of Fn Cpf1.Methods1.The genome of novicida subspecies was used as the template.The FnCpf1 gene was amplified by PCR and subcloned into prokaryotic expression vector pET-32a(+),and the recombinant plasmid pET32a(+)-FnCpf1 was verified by restriction digestion and sequencing.2.The pET-32a(+)-FnCpf1 was transformed into E.coli BL21 and the recombinant protein FnCpf1 was induced with IPTG.The FnCpf1 protein was purified by the nickel ion chelating beads affinity purification.3.FnCpf1 polyclonal antibody was prepared by immunization of New Zealand white rabbits,and the antiserum titer and specificity of the antibody was measured by indirect ELISA and Western blot respectively.4.To construct an inactivated mCherry fluorescent reporter vector,and transform it into the competent state of E.coli expressing the FnCpf1 protein,and then the red colonies on the solid medium could be observed directly to verify the genome of vector was cut and recombined.Results1.The size of restriction fragment of recombinant plasmid was 3900 bp.Restriction and sequencing analysis proved that recombinant plasmid pET32a(+)-FnCpf1 was constructed successfully.2.The expression of FnCpf1 protein was obtained by affinity chromatography on Ni-ion chelate beads.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 protein was more than 95% and its concentration was 1.4mg/ml.3.Immunized New Zealand White Rabbit with purified protein 3 times to preparation of polyclonal antibody.ELISA showed the titer of anti-FnCpf1 antiserum was 1: 512000,and Western blot test demonstrated that the prepared antibody could specifically bind to FnCpf1 protein.4.The mCherry red fluorescent reporter gene which inserted an inactivation sequence was transformed into E.coli expressing FnCpf1 and then we could see there were red colonies on the solid medium.But the negative control was not.Conclusions1.FnCpf1 recombinant expression vector was successfully constructed and the recombinant protein of FnCpf1 was induced,expressed and purified in prokaryotic system.2.FnCpf1 polyclonal antibody was prepared with high titer and specificity,which would lay the foundation for further study on the biological characteristics of FnCpf1 protein.3.In the expression of FnCpf1 E.coli,the mutated mCherry red fluorescent reporter gene was targeted and cleaved by CRISPR-Cpf1 system,and the nuclease activity of FnCpf1 protein was proved.