Expression, Biological Activity Analysis And Polyclonal Antibody Preparation Of Human Cofilin-1

Objective:To construct the recombinant plasmid of pET-28a(+)-cofilin-1, and express and purify the recombinant protein of cofilin-1, and prepare specific polyclonal antibody of Mdfic protein.Methods and Results:Cofilin-1 gene cDNA was amplified by RT-PCR from human cervical cancer cell. The cofilin-1 cDNA was cloned into pET28a(+) plasmid which has a 6×His tag, and the restriction enzyme cutting site of sense primer and antisense primer was Nde I and Xho I respectively. The recombinant plasmid was transformed into competence Escherichia coli DH5a. The positive recombinant clones were identified by restriction endonuclease digestion and sequencing. Then the positive recombinant plasmid was transformed into competence Escherichia coli BL21 (DE3). The recombinant protein was analysis by SDS-PAGE and Western blot assay after the recombinant Escherichia coli BL21 was induced by IPTG. Optimum temperature, IPTG concentration and induction time was determined for the highest level of recombinant protein. The results showed that when OD600 was 0.6～0.8, after being induced with 1.0 mM/L IPTG at 20℃for 20 hours, the expression level of recombinant protein was highest. The recombinant protein cofilin-1 was expressed in soluble form, and the best yieds reached 33% of total protein in E.coli.The recombinant proten cofilin-1 was purified by Ni-NTA His Bind Resins column.And then the biological activity of recombinant protein was analysised by non-denatured PAGE. The recombinant protein cofilin-1 could bind to binary complex actin-DNase I and formed ternart complex cofilin-1-actin-DNase.Anti-cofilin-1 polyelonal antibody was prepared by immunizing rabbits with purified recombinant protein cofilin-1. The titer of the polyclonal antibody against cofilin-1 was about 1:128000 by ELISA. After being purified by salting out and anion-exchange chromatography, the titer of the polyclonal antibody was about 1:64000. The Anti-cofilin-1 polyelonal antibody had high specificity by Western blot assay.Conclusions:The recombinant protein cofilin-1 which had activity and the Anti-cofilin-1 polyelonal antibody which had high titer and specificity were successfully prepared.