结核分枝杆菌早期分泌蛋白MPT64的原核表达及其在结核病血清学诊断上的初步应用  

Prokaryotic expression of early secretory protein MPT64 from Mycobacterium tuberculosis and its application in serological diagnosis of tuberculosis

作　　者：屈蓉 吴康[2] 吴娟[2] 范小勇 吕建新[1] QU Rong;WU Kang;WU Juan;FAN Xiao-yong;LYU Jian-xin(School of Laboratory,Medicine and Life Science,Wenzhou Medical University,Wenzhou 325035,Zhejiang Province,China;不详)

机构地区：[1]温州医科大学检验医学院、生命科学学院,浙江温州325035 [2]上海市(复旦大学附属)公共卫生临床中心,上海201508

出　　处：《中国生物制品学杂志》2021年第5期566-570,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金项目(31771004);“十三五”传染病重大专项(2018ZX10302301);上海市科委项目(19XD1403100,20Y11903400)。

摘　　要：目的原核表达、纯化结核分枝杆菌(Mycobacterium tuberculosis,Mtb)早期分泌蛋白MPT64,并初步评估其在结核病(tuberculosis,TB)血清学诊断中的价值。方法PCR扩增Mtb标准菌株H37Rv mpt64基因,克隆至原核表达载体pET28a(+),构建重组质粒pET28a-mpt64,转化大肠埃希菌BL21(DE3)后,进行重组蛋白的原核诱导表达及纯化。将600μg纯化的MPT64蛋白经皮下多点免疫新西兰大白兔,隔周免疫1次,共4次,末次免疫2周后,耳缘静脉采血分离血清,ELISA法检测血清效价,Western blot法鉴定抗原特异性。收集44例TB确诊患者(TB组)和36名健康对照人群[HC(healthy control)组]血清,间接ELISA法检测血清中MPT64抗原的特异性IgG、IgA抗体水平,抗原对照为Gro ES和Ag85A。结果重组表达质粒pET28a-mpt64经双酶切鉴定及测序证明构建正确;表达的重组MPT64蛋白主要以包涵体形式存在,部分以可溶性形式存在,经Ni2+-NTA亲和纯化后,纯度可达90%,蛋白浓度约2.0 mg/m L;MPT64兔多克隆抗血清效价达1∶2048000,纯化的MPT64蛋白可与其发生反应,在相对分子质量约24000处可见特异性蛋白条带;TB组血清中MPT64抗原特异性IgG和IgA水平均显著高于HC组(P<0.05),而Gro ES、Ag85A抗原特异性IgA和IgG水平差异无统计学意义(P>0.05)。结论重组MPT64蛋白具有较好的血清学诊断效能,可作为TB血清学早期诊断的候选抗原之一。Objective To express the early secretory protein MPT64 from Mycobacterium tuberculosis(Mtb) in prokaryotic cells,purify the recombinant protein and evaluate its significance in serological diagnosis of tuberculosis(TB).Methods The mpt64 gene was amplified from the genome of Mtb standard strain H37 Rv by PCR and cloned into prokaryotic expression vector pET28 a(+). The constructed recombinant plasmid pET28 a-mpt64 was transformed to E. coli BL21(DE3) for expression under induction of IPTG. The expressed recombinant protein was purified by Ni2+-NTA chromatography,with which New Zealand white rabbits were immunized s. c. at a dosage of 600 μg in several sites,every other week for 4 times. Two weeks after the last immunization,blood samples were collected from ear vein,from which sera were separated,determined for antibody titer by ELISA,and identified for antigen specificity by Western blot. Serum samples were collected from 44 patients with TB and 36 healthy volunteers and determined for IgG and IgA levels against MPT64 by indirect ELISA,using Gro ES and Ag85 A as control antigens. Results Both restriction analysis and sequencing proved that recombinant plasmid pET28 a-mpt64 was constructed correctly. The recombinant MPT64 protein was mainly expressed in the form of inclusion body and partly in soluble form,of which the purity reached 90% and the protein concentration was about 2. 0 mg/m L after purification by Ni2+-NTA chromatography. The rabbit polyclonal antiserum against MPT64 reached a titer of 1 ∶ 2 048 000,which showed reaction with the purified MPT64 protein and formed a specific protein band with relative molecular mass of about 24 000. Both the IgG and IgA levels against MPT64 in patients with TB were significantly higher than those in healthy volunteers as control(P < 0. 05),while those against Gro ES and Ag85 A showed no significant difference(P > 0. 05). Conclusion Recombinant MPT64 showed good efficiency in serological diagnosis of TB,which might be used as a candidate antigen for early serological

关 键 词：结核分枝杆菌 MPT64 免疫学诊断 血清学诊断 

分 类 号：R378.912[医药卫生—病原生物学]