Cloning And Expression Of Human Cystatin C And Preparation Of Its Monoclonal/Polyclonal Antibody

Objective: To construct a prokaryotic plasmid to express the human Cystatin C recombinant protein and obtain anti-Cystatin C mAb/pAb by immunizing mice/rabbit, and to identify the biological properties of the mAb/pAb. We prepared this project to detect the expression, distribution of human Cystatin C in stomach neoplasms and esophageal cancer tissues.Methods: The coding sequence of human Cystatin C was amplified by RT-PCR from human gastric cancer AGS cell and cloned into the vector pET44, and then it was expressed in the E. coli BL 21(DE3) to get Cystatin C recombinant protein. The refolding denatured purified Cystatin C recombinant protein was injected to immunize the BALB/C mice to develop anti-Cystatin C mAb, and immunized the rabbit to develop anti-Cystatin C pAb. Splenocytes of the immunized mice were collected and fused with the mouse myeloma cell line NS-1 cells. ELISA, Western blot and were used to detect specificity of mAb/pAb. The expression of Cystatin C in neoplasms and esophageal cancer tissues was investigated by immunohistochemistry and RT-PCR.Results: The prokaryotic plasmid expressing the recombinant protein was constructed, and the Cystatin C recombinant protein was expressed and purified. The subtype of the mAb were IgG1. Westem blot and ELISA analysis indicated the mAb/pAb showed specific combination with Cystatin C protein. Cystatin C was strongly expressed in stomach neoplasms and esophageal cancer tissues compared to adjacent normal tissues. The mRNA of Cystatin C was also higher expressed in tumors than in normal tissues.Conclusion: The Cystatin C recombinant protein is highly purified and has strong antigenicity. The Cystatin C and its mAb/pAb are produced successfully. Cystatin C and its Mrna were strongly expressed in stomach neoplasms and esophageal cancer tissues compared to adjacent normal tissues.