CAJ | 학술논문

目的获得重组MPT64蛋白，研究其免疫学特性，评价其在结核病血清学诊断中的价值。方法应用基因工程技术表达、纯化MPT64蛋白，通过Western blotting分析其抗原性。分别以结核分枝杆菌PPD或纯化的rMPT64蛋白为抗原，通过ELISA方法及结核病一步法检测血清中抗结核抗体。结果重组质粒pET 15b-MPT64测序表达除83位密码子由CCA突变为CCG外，其余密码子均与报道的相同，但其氨基酸序列无变化。它在大肠杆菌BL21(DE3)细胞内以包涵体形式存在，表达量占菌体总蛋白的20～30%，分子量约26kDa，Western blotting分析它与抗结核分枝杆菌多克隆抗体具有良好的免疫反应性。纯化后的rMPT64样品经SDS-PAGE和光密度扫描分析表明其纯度约为80%左右，每100ml培养菌可获得10mg左右的重组蛋白。以33例正常人血清的OD值+2S为正常界限值，PDD的特异性和敏感性分别为94%(31/33)、63.7%(21/33)、66.7%；rMPT64纯化蛋白分别为 94%(31/33)、30.3(10/33)；一步法分别为88%(29/33)、66.7%(22/23)。结论 pET15b-MPT64大肠杆菌工程菌能以包涵体形式高效表达重组MPT64蛋白，该蛋白具有较高的抗原特异性和免疫反应性。rMPT64纯化蛋白有可能成为结核病血清学诊断的组合抗原之一。
목적획득중조MPT64단백，연구기면역학특성，평개기재결핵병혈청학진단중적개치。방법응용기인공정기술표체、순화MPT64단백，통과Western blotting분석기항원성。분별이결핵분지간균PPD혹순화적rMPT64단백위항원，통과ELISA방법급결핵병일보법검측혈청중항결핵항체。결과중조질립pET 15b-MPT64측서표체제83위밀마자유CCA돌변위CCG외，기여밀마자균여보도적상동，단기안기산서렬무변화。타재대장간균BL21(DE3)세포내이포함체형식존재，표체량점균체총단백적20～30%，분자량약26kDa，Western blotting분석타여항결핵분지간균다극륭항체구유량호적면역반응성。순화후적rMPT64양품경SDS-PAGE화광밀도소묘분석표명기순도약위80%좌우，매100ml배양균가획득10mg좌우적중조단백。이33례정상인혈청적OD치+2S위정상계한치，PDD적특이성화민감성분별위94%(31/33)、63.7%(21/33)、66.7%；rMPT64순화단백분별위 94%(31/33)、30.3(10/33)；일보법분별위88%(29/33)、66.7%(22/23)。결론 pET15b-MPT64대장간균공정균능이포함체형식고효표체중조MPT64단백，해단백구유교고적항원특이성화면역반응성。rMPT64순화단백유가능성위결핵병혈청학진단적조합항원지일。
Objective　To obtain the recombinant MPT64 protein,to study its immunological characteristics,and to evaluate its potential value fo r serodiagnosis of tuberculosis.Methods　The gene coding MPT64 prote in inserted into a expression vector pET-15b,and then transferred to E.coli BL21(DE3).The expressed product was purified by metal chelation chromatograph y,and analyzed its immunogenicity by western blotting.66 human serum was detecte d 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 t hat 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 amo unted to 20～30% of total bacterial proteins.Its molecular weight was about 26 k ilodalton.It can produce good immunoreactivity with serum from goats vaccinated with M.tuberculosis by western blotting.The purity of final product was abou t 80%.10mg of rMPT64 protein per 100ml culture could be obtained.The positive cu toff values were OD492 plus 2 standard deviation of 33 negative serum dete cted by ELISA.Of 33 serum from tuberculosis patients,the specificity and sensiti vity of PPD as antigen of ELISA were 94% and 63.7%,That of rMPT64 were 94% and 3 0.3%,and that of tuberculosis one-step test were 88% and 66.7%,respectively.Conclusion　The recombinant MPT64 protein could be highly expres sed in the form of inclusion bodies in E.coli.It had good antigenic specific ity and immunoreactivity,and might be selected as one of serodignostic antigen o f tuberculosis.