Cloning and expression of MICB gene and its application for the detection of anti-MICB antibodies

Abstract

Objective To construct a prokaryotic expression system for major histocompatibility complex class Ⅰ chain-related gene B (MICB) and to establish an ELISA method for the detection of anti-MICB antibodies in patients with kidney transplantation. Methods The MICB cDNA fragments were obtained by RT-PCR with a pair of specific primers. The MICB cDNA and the prokaryotic expression vector pET-28a were digested by two restriction enzymes and ligated to construct the recombinant expression plasmid pET-28a-MICB. The transformed E. coli BL21 DE3 strains carrying recombinant expression plasmid were induced by IPTG to express MICB protein. The expressed recombinant proteins were identified by Western blot assay and purified by Ni-NTA Spin column. The purified proteins were coupled to ELISA for the detection of anti-MICB antibodies in patients with kidney transplantation. Results Three common MICB fragments contained the exons 2 and 3 were obtained. The recombinant proteins were expressed in E. coli BL21 DE3 strains carrying pET-28a-MICB and successfully purified by the Ni-NTA Spin column. Results of the Western blot assay confirmed that the obtained proteins were the target proteins. The ELISA method was successfully established and used for the detection of anti-MICB antibodies in 24 patients with kidney transplantation. The absorbance values indicated that the sensitivities of three recombinant MICB proteins were different. Conclusion The expression system for MICB gene was successfully constructed. The established ELISA for the detection of anti-MICB antibodies would pave the way for further investigation on the correlations between MICB protein and transplantation immunity. Key words: MICB; Protein expression and purification; Antibody detection