Gene cloning and polyclonal antibody preparation of human PIWI4 and preliminary application

Abstract

Objective To generate rabbit polyclonal antibody against human PIWIL4 protein, to identify its functional characterization, measure differential expression and determine the cellular localization of PIWIL4 protein in various cell lines. Methods Prokaryotic expressed plasmid pGEX-5X-1-PIWIL4 was constructed and transformed to E. coli BL21 to induce expression by IPTG. The fusion protein was injected into rabbits subcutaneously to produce polyclonal antibodies after purification by gel regaining. Enzyme linked im-munosorbent assay (ELISA) was operated to detect the titer of the antibodies, western blotting was used to identify the specificity and sensitivity of the antibodies and detect the differential expression of PIWIL4 protein in various cell lines. Meanwhile, immunofluorescence experiments were used to show cellular localization of PIWIL4 protein. Results The prokaryotic expressed plasmid was constructed correctly. PIWIL4 protein was expressed and purified, and then rabbit polyclonal antibodies against PIWIL4 were generated after immunization with the fusion protein. The titer detected by ELISA was 1:20 000. The evidence of western blotting demonstrated the specificity of the antibodies. Finally, we successfully observed the differential expression and cellular localization of PIWIL4 protein in various cell lines. Conclusion The polyclonal antibody against PIWIL4 protein has been achieved successfully. It will be propitious for the intensive functional study of PIWIL4. Key words: PIWIL4; Prokaryotic expression; Antibody; Cancer