Abstract:

Objective To perform the prokaryotic expression,purification and identification of Cystatin of Cyprinus carpio var.Jian,and to further prepare a polyclonal antibody targeting Cystatin as the antigen. Methods The E.coli　BL21（DE3）,in which the pET-30a-Cystatin plasmid had been transferred,was induced by IPTG to express the recombinant protein,which was purified with Ni2+-NTA affinity chromatography.Then the purity of recombinant Cystatin and inhibitory activity was identified using high performance liquid and Azocasein respectively.The QuickAntibody-Mouse5W fast adjuvant and purified recombinant Cystatin were used to immunize Balb/C mice to get antiserum,whose titer was identified with sandwich ELISA and whose specificity  to recognize antigens was assessed with Western blotting and immunohistochemistry. Results The molecular weight of interest protein of prokaryotic expression was approximately 20 000.After Ni2+-NTA affinity chromatography,the recombinant Cystatin was shown as a single band on the SDS-PAGE and its purity was above 95%.Azocasein assay showed that 50% activityof papain was inhibited by the recombinant Cystatin of 0.014 μg.ELISA showed Cystatin antiserum with titer of 1∶512 000 was obtained.The results of Western blotting showed the Cystatin was absence in the total protein of E.coli　BL21(DE3) with transferred plasmid pET-30a,in contrast with transferred plasmid pET-30a-Cystatin and the sample in every process of purifying,the Cystatin was presence and showed a single band,and immunohistochemistry indicated the different tissues of Cyprinus carpio var.Jian showed different positive staining,the antiserum could specifically bind to the Cystatin expressed in both prokaryotic and eukaryotic systems. Conclusion The recombinant Cystatin of Cyprinus carpio var.Jian is expressed and purified successfully.And finally,the antiserum with high titer polyclonal antibody recognizing Cystatin of Cyprinus carpio var.Jian is obtained,which owns the high specificity and sensitivity.

Key words: Cystatin, prokaryotic expression, polyclonal antibody