Cloning, Expression And The Monoclonal Antibody Preparation Of Procalcitonin

Objective: To synthetize the whole gene of procalcitonin, to express the fusionprotein, and to prepare the monoclonal antibody of procalcitonin, then to provid thesubject for study of the origin,pathological and physiological functions ofprocalcitonin.Methods: Human procalcitonin gene was reconditioned some rare codons but itsamino acid sequence was retained invariably, so as to the fusion protein ofprocalcitonin could express high performance. Eighteen primers were designed byusing the computer software. By using single-step assembly of a gene and entireplasmid from large numbers of oligodeoxyribonucleotides technique, the whole geneof procalcitonin was synthetized, then inserted into the prokaryotic expression vectorpGEX-4T-1, the recombinant plasmid was transformed into E. coli and expressed therecombinant protein. The recombinant protein and the monoclonal antibody ofprocalcitonin were assessed with western blot The fusion protein was purifiedthrough GSTrapFF column for immunizing Balb/c mouse. Preparing the monoclonalantibody was followed with the traditional method. The specificity of the monoclonalantibody was identified with the antigen of procalcitonin.Results: The whole gene of procalcitonin was synthetized successfully. And the DNA was inserted into the prokaryotic expression vector pGEX-4T-1 and expressedthe recombinant protein. The recombinant protein and the monoclonal antibody ofprocalcitonin were assessed with western blot. Western blot analysis showed thefusion protein could react with the monoclonal antibody of procalcitonin specifically.A high level expression of GST-PCT was obtained and followed by purification withmore than 60 percent. The purification of protein were used as immunogen to preparethe monoclonal antibody by using the traditional method. The titer of monoclonalantibody were detected by indirect ELISA and two monoclonal antibody wereobtained with titer 10^-5 and 10^-6 respectively. The monoclonal antibody and theantigen of procalcitonin were identified with western blot. Western blot analysisshowed the monoclonal antibody of procalcitonin could react with the antigenspecifically.Conclusion: 1. We reconditioned some rare codons of Human procalcitoningene and retained its amino acid sequence invariably, so as to the fusion protein ofprocalcitonin could express high performance. 2. We have successfully synthetizedthe whole gene of procalcitonin by using single-step assembly of a gene and entireplasmid from large numbers of oligodeoxyribonucleotides technique and havesuccessfully gained the recombinant protein. There provided the subject forpreparation of the monoclonal antibody of procalcitonin. 3. We have successfullygenerated a monoclonal antibody of human procalcitonin with the traditional methodand will contribute to functional study of procalcitonin further. The result has thesignificant deviation. It is suggested that procalcitonin act as a marker for prognosisevaluation for severe bacterial infections.