CAJ | 학술논문

目的：在大肠杆菌中表达结核分枝杆菌RD2区域的NrdF1、PE_PGRS35、Rv1985c和Rv1986蛋白，并评价诊断牛结核病中的应用潜能。方法以结核分枝杆菌 H37Rv基因组 DNA 为模板，PCR 扩增 nrdF1，pe_p grs35，rv1985c 和rv1986基因，并将其克隆到表达载体中，重组表达质粒转化 E ．coli BL21（DE3），IPTG诱导目的蛋白表达，镍柱亲和纯化重组蛋白，SDS-PAGE和 Western blotting 试验鉴定目的蛋白的表达及其反应原性。以纯化的重组蛋白作为包被抗原，通过ELISA方法测定牛血清中特异性抗体，以之评价这些蛋白用于牛结核病抗体检测的价值。结果成功表达了 NrdF1、PE_PGRS35、Rv1985c和Rv1986蛋白，相对分子质量为42 ku、63 ku、46 ku和41 ku ，对表达产物进行镍柱亲和纯化，获得了纯度较高的融合蛋白。重组蛋白均能与抗 HIS单抗发生特异反应，表现出良好的反应原性。通过间接ELISA方法检测牛血清中特异性抗体，阳性检出率分别为7．35％、22．06％、16．18％和16．18％。结论结核分枝杆菌原核表达的 N rdF1、PE_PGRS35、Rv1985c和Rv1986蛋白具有用作牛结核病血清学诊断试剂的潜能。
목적：재대장간균중표체결핵분지간균RD2구역적NrdF1、PE_PGRS35、Rv1985c화Rv1986단백，병평개진단우결핵병중적응용잠능。방법이결핵분지간균 H37Rv기인조 DNA 위모판，PCR 확증 nrdF1，pe_p grs35，rv1985c 화rv1986기인，병장기극륭도표체재체중，중조표체질립전화 E ．coli BL21（DE3），IPTG유도목적단백표체，얼주친화순화중조단백，SDS-PAGE화 Western blotting 시험감정목적단백적표체급기반응원성。이순화적중조단백작위포피항원，통과ELISA방법측정우혈청중특이성항체，이지평개저사단백용우우결핵병항체검측적개치。결과성공표체료 NrdF1、PE_PGRS35、Rv1985c화Rv1986단백，상대분자질량위42 ku、63 ku、46 ku화41 ku ，대표체산물진행얼주친화순화，획득료순도교고적융합단백。중조단백균능여항 HIS단항발생특이반응，표현출량호적반응원성。통과간접ELISA방법검측우혈청중특이성항체，양성검출솔분별위7．35％、22．06％、16．18％화16．18％。결론결핵분지간균원핵표체적 N rdF1、PE_PGRS35、Rv1985c화Rv1986단백구유용작우결핵병혈청학진단시제적잠능。
Proteins encoded by regions of difference (RD) of Mycobacterium tuberculosis (MTB) constitute a potential source of specific antigens for vaccine development and immunodiagnosis .In the present study ,four genes named nrdF1 , pe_pgrs35 ,rv1985c ,and rv1986 from RD2 of MTB were cloned and overexpressed in E .coli with the induction of IPTG .Western blotting assay showed that these recombinant fusion proteins could well react with anti-His tag monoclonal antibody ,which in-dicated their good immunoreactivity .The serodiagnosis potential applications of these four proteins in bovine tuberculosis were further evaluated .An indirect ELISA assay was established by using fusion proteins as coating antigens for detection of their specific antibodies in bovine sera .The positive rates were 7 .35% in NrdF1 ,22 .06% in PE_PGRS35 ,16 .18% in Rv1985c and 16 .18% in Rv1986 respectively .All the results suggest that these fusion proteins have the potential application in serodiagnosis of bovine tuberculosis .