关键词: 抗原, MPT64蛋白, 基因表达, 血清学诊断, 分枝杆菌,结核

Abstract: Objective To obtain the recombinant MPT64 protein,to study its immunological characteristics,and to evaluate its potential value for serodiagnosis of tuberculosis.Methods The gene coding MPT64 protein inserted into a expression vector pET 15b,and then transferred to E.coli BL21(DE3).The expressed product was purified by metal chelation chromatography,and analyzed its immunogenicity by western blotting.66 human serum was detected the antibodies against M.tuberculosis rMPT64 and PPD antigens by ELISA,and antibodies against M.tuberculosis antigens by tuberculosis one step test.Results Sequencing recombinant plasmid pET 15b MPT64 showed that there was a mutation (CCA→CCG) at codon 83,but its amino acid had no change.The recombinant MPT64 protein existed in inclusion bodies of E.coli ,and amounted to 20～30% of total bacterial proteins.Its molecular weight was about 26 kilodalton.It can produce good immunoreactivity with serum from goats vaccinated with M.tuberculosis by western blotting.The purity of final product was about 80%.10mg of rMPT64 protein per 100ml culture could be obtained.The positive cutoff values were OD₄₉₂ plus 2 standard deviation of 33 negative serum detected by ELISA.Of 33 serum from tuberculosis patients,the specificity and sensitivity of PPD as antigen of ELISA were 94% and 63.7%,That of rMPT64 were 94% and 30.3%,and that of tuberculosis one step test were 88% and 66.7%,respectively.Conclusion The recombinant MPT64 protein could be highly expressed in the form of inclusion bodies in E.coli. It had good antigenic specificity and immunoreactivity,and might be selected as one of serodignostic antigen of tuberculosis.

Key words: Antigen, MPT64 protein, Gene expression, Serodiagnosis, Mycobacterium tuberculosis