Prokaryotic Expression Of Human CTnI And CTnI–linker-TnC Fusion Protein And Preparation Of Monoclonal Antibodies Against CTnI

AIM: Prokaryotic expression of human cardiac troponin I(c Tn I) and instead of the natural proteins. And using them to immune the Balb/c mouse, prepare several c Tn I-specific monoclonal antibodies(m Abs) with high specificity and affinity, laying a foundation of c Tn I detection method.METHOD: A short DNA sequence which codes 15 neutral amino acid residues was used to link c Tn I and c Tn C. And acquired the purpose genes of c Tn I and c Tn C through PCR technics.Then, the recombinant prokaryotic expression E.coli BL21 cells were constructed which contained the aim genes of c Tn I, c Tn C and c Tn I-linker-Tn C and expressed the purpose proteins. We used c Tn I-linker-Tn C and the natural c Tn I to immunize the six week olds Balb/c mouse to prepare the m Abs of c Tn I by using the classic hybridoma fusion method. Identified and analyzed the affinity and specificity of the ascites monoclonal antibodies.RESULTS: Constructed recombinant prokaryotic expression E.coli BL21 cells of purpose proteins c Tn I, c Tn C and c Tn I-linker-Tn C successfully and achieved the high soluble expression of them. And they all reserved their immunogenicity well after identification with several methods. It proved the prokaryotic expression proteins may instead of the natural ones. A total of 8 m Abs were prepared after immunized with natural c Tn I and the titer of 6H7 and 4A8 can reach 1:40 thousand. The rate of the positive holes reached 30% after immunized with c Tn I-linker-Tn C and there were 18 holes` OD values were above 2.0. And the following work is undergoing.CONSULUTION: We achieved prokaryotic expression of c Tn I, c Tn C and c Tn I-linker-Tn C successfully. They all reserved their immunogenicity well and may be used for instead of the natural ones in the future. We have prepared several m Abs of c Tn I successfully and lay the foundation of c Tn I detection method.