CAJ | 학술논문

目的构建结核分枝杆菌H37Rv 株esat-6-cfp-10(简称ec)融合基因及其原核表达载体pET-23b(+).esat-6-cfp-10[以下简称pET-23b(+)-ec],表达、纯化融合蛋白rESAT-6-CFP-10(简称rEC),并分析其免疫反应性.方法采用基因拼接技术将ESAT-6和CFP-10编码基因用疏水甘氨酸接头(Gly4Ser)3进行PCR扩增融合.构建TA克隆载体pMD 19-T-esat-6-cfp-10(简称pMD+ 19-T-ec),利用PCR、酶切和测序进行鉴定.经限制性内切酶Nde Ⅰ、Xho Ⅰ双酶切后将融合基因定向克隆入原核表达载体pET-23b(+).将构建正确的重组质粒pET-23b(+).ec转化E.coli BL21(DE3)pLysE,异丙基-β-D-硫代半乳糖苷(IPTG)诱导rEC的表达.十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)和Westem印迹法分析其表达情况.用镍离子螯合亲和层析柱纯化融合蛋白.用Western印迹初步评价融合蛋白的免疫反应性.结果融合基因及其原、核表达载体构建成功,融合蛋白以可溶性非包涵体形式表达,相对分子质量为21 000,表达量约占菌体总蛋白的35%.经亲和层析后得到了纯度达97.2%的融合蛋白.Western印迹证实,融合蛋白能与确诊的肺结核患者血清发生特异性免疫应答.结论成功构建了原核表达载体pET-23b(+).ec,获得了rEC融合蛋白,为rEC融合蛋白在结核病诊断中的应用奠定了基础.
목적 구건결핵분지간균H37Rv 주esat-6-cfp-10(간칭ec)융합기인급기원핵표체재체pET-23b(+).esat-6-cfp-10[이하간칭pET-23b(+)-ec],표체、순화융합단백rESAT-6-CFP-10(간칭rEC),병분석기면역반응성.방법 채용기인병접기술장ESAT-6화CFP-10편마기인용소수감안산접두(Gly4Ser)3진행PCR확증융합.구건TA극륭재체pMD 19-T-esat-6-cfp-10(간칭pMD+ 19-T-ec),이용PCR、매절화측서진행감정.경한제성내절매Nde Ⅰ、Xho Ⅰ쌍매절후장융합기인정향극륭입원핵표체재체pET-23b(+).장구건정학적중조질립pET-23b(+).ec전화E.coli BL21(DE3)pLysE,이병기-β-D-류대반유당감(IPTG)유도rEC적표체.십이완기류산납취병희선알응효전영(SDS-PAGE)화Westem인적법분석기표체정황.용얼리자오합친화층석주순화융합단백.용Western인적초보평개융합단백적면역반응성.결과 융합기인급기원、핵표체재체구건성공,융합단백이가용성비포함체형식표체,상대분자질량위21 000,표체량약점균체총단백적35%.경친화층석후득도료순도체97.2%적융합단백.Western인적증실,융합단백능여학진적폐결핵환자혈청발생특이성면역응답.결론 성공구건료원핵표체재체pET-23b(+).ec,획득료rEC융합단백,위rEC융합단백재결핵병진단중적응용전정료기출.
Objective To construct esat-6-cfp-10 fusion gene (ec) and its expression vector pET-23b( + )-esat-6-cfp-10 [pET-23b( + )-ec], and express rESAT-6-CFP-10 (rEC)fusion protein of Mycobacterium tuberculosis (MTB) H37Rv and evaluate its immunoreactivity. Methods Fused gene encoding ESAT-6 and OP-10 of MTB was linked with the (Gly4Ser)3 linker by gene SOEing. Construction of TA cloning vector pMDR 19-T-esal-6-cfp-10 (pMDR 19-T-ec) was identified by PCR, restriction analysis and sequencing. After pMDR 19-T-ec was digested with double restriction enzymes (Nde Ⅰ and Xho Ⅰ ), the fused gene was inserted into prokaryotic expression vector pET-23b ( + ). The recombinant plasmid pET-23b( + )-ec was transformed into E. coli BL21(DE3) pLysE, and IPTG was used to induce the expreestion of rEC. Its expression was detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. rEC was purified by nickel-chelate affinity chromatography. The immunoreactivity of rEC was preliminarily evaluated by Western blot. Results A recombinant plasmid pET-23b( + )-ec was successfully constructed and could stably express a 21 000 rEC, which accounted for 35% total proteins of bacillus. It existed in soluble form in E. coli. After purification, the purity of rEC reached 97.2% . Its immunoreactivity was confirmed by Western blot. Conclusions The prokaryotic expression vector pET-23b( + )-ec has been constructed and the fusion protein rEC has been obtained successfully, which provides an experimental basis for the application of rEC in the diagnosis of tuberculosis.