安徽医科大学学报
安徽醫科大學學報
안휘의과대학학보
Acta Universitatis Medicinalis Anhui
2015年
10期
1404-1409
,共6页
周鹏%丁莹莹%林子玉%冯娇娇%高彩霞%王锦红%杨华%温宗梅%潘卫%邓松华
週鵬%丁瑩瑩%林子玉%馮嬌嬌%高綵霞%王錦紅%楊華%溫宗梅%潘衛%鄧鬆華
주붕%정형형%림자옥%풍교교%고채하%왕금홍%양화%온종매%반위%산송화
结核分枝杆菌%Rv3388%ELISA%特异性%敏感性
結覈分枝桿菌%Rv3388%ELISA%特異性%敏感性
결핵분지간균%Rv3388%ELISA%특이성%민감성
Mycobacterium tuberculosis%Rv3388%ELISA%specificity%sensitivity
目的 对结核分枝杆菌( MTB) PE家族的所有成员的结构进行分析,预测其抗原表位,截取Rv3388蛋白的优势抗原片段,对重组Rv3388蛋白及其它6种MTB特异性抗原进行评估,探讨不同结核特异性抗原与血清抗体的反应模式,评价血清学检测在结核病诊断中的价值. 方法 利用基因合成技术重叠延伸PCR扩增Rv3388蛋白637~731位的编码基因片段, 原核表达并纯化重组蛋白pET32a/Rv3388637-731 ,将纯化的重组蛋白免疫BALB/c小鼠,采用间接ELISA法对该抗血清进行免疫原性分析. 同时对这7种MTB特异性蛋白的特异性及敏感性进行评价. 结果 重组蛋白pET32a/Rv3388637-731在大肠杆菌中的表达量占全菌蛋白的80%,ELISA显示有较强的免疫原性. 7 种 MTB特异性抗原具有不同的反应模式,单个抗原检测敏感性较差. 结论 对MTB PE家族的蛋白结构分析,表达并纯化重组蛋白pET32a/ Rv3388637-731 ,7种蛋白在血清抗体检测中具有抗原互补性,不同抗原与机体反应存在不同反应模式,提高结核抗体检测敏感性应多种抗原联合检测.
目的 對結覈分枝桿菌( MTB) PE傢族的所有成員的結構進行分析,預測其抗原錶位,截取Rv3388蛋白的優勢抗原片段,對重組Rv3388蛋白及其它6種MTB特異性抗原進行評估,探討不同結覈特異性抗原與血清抗體的反應模式,評價血清學檢測在結覈病診斷中的價值. 方法 利用基因閤成技術重疊延伸PCR擴增Rv3388蛋白637~731位的編碼基因片段, 原覈錶達併純化重組蛋白pET32a/Rv3388637-731 ,將純化的重組蛋白免疫BALB/c小鼠,採用間接ELISA法對該抗血清進行免疫原性分析. 同時對這7種MTB特異性蛋白的特異性及敏感性進行評價. 結果 重組蛋白pET32a/Rv3388637-731在大腸桿菌中的錶達量佔全菌蛋白的80%,ELISA顯示有較彊的免疫原性. 7 種 MTB特異性抗原具有不同的反應模式,單箇抗原檢測敏感性較差. 結論 對MTB PE傢族的蛋白結構分析,錶達併純化重組蛋白pET32a/ Rv3388637-731 ,7種蛋白在血清抗體檢測中具有抗原互補性,不同抗原與機體反應存在不同反應模式,提高結覈抗體檢測敏感性應多種抗原聯閤檢測.
목적 대결핵분지간균( MTB) PE가족적소유성원적결구진행분석,예측기항원표위,절취Rv3388단백적우세항원편단,대중조Rv3388단백급기타6충MTB특이성항원진행평고,탐토불동결핵특이성항원여혈청항체적반응모식,평개혈청학검측재결핵병진단중적개치. 방법 이용기인합성기술중첩연신PCR확증Rv3388단백637~731위적편마기인편단, 원핵표체병순화중조단백pET32a/Rv3388637-731 ,장순화적중조단백면역BALB/c소서,채용간접ELISA법대해항혈청진행면역원성분석. 동시대저7충MTB특이성단백적특이성급민감성진행평개. 결과 중조단백pET32a/Rv3388637-731재대장간균중적표체량점전균단백적80%,ELISA현시유교강적면역원성. 7 충 MTB특이성항원구유불동적반응모식,단개항원검측민감성교차. 결론 대MTB PE가족적단백결구분석,표체병순화중조단백pET32a/ Rv3388637-731 ,7충단백재혈청항체검측중구유항원호보성,불동항원여궤체반응존재불동반응모식,제고결핵항체검측민감성응다충항원연합검측.
Objective To analyze the structure of all members in the PE family of Mycobacterium tuberculosis ( MTB)and predict the antigen epitope, we chose the dominant antigen fragments of Rv3388 protein and estimate the anti-genicity of not only the recombinant Rv3388 protein but aslo the other six kinds of specific antigens of MTB. To in-vestigate the reaction model between the different tuberculosis specific antigen and serum antibody, and to evaluate the value of serological detection in the diagnosis of tuberculosis. Methods The gene coding 637~731 amino acid fragment of Rv3388 was amplified by over-lap extension-PCR. The prokaryotic expressed and purified recombinant protein pET32a/Rv3388637-731 was utilized to immunize BALB/c mice, and the immunogenicity of the antiserum and the specificity and sensitivity of seven kinds specific proteins of MTB were analyzed by indirect ELISA. Results The amount of recombinant protein pET32a/Rv3388637-731 expressed in E. coli was 80% of the total protein. The results of ELISA showed strong immunogenicity. The reaction patterns of antigens were different with each oth-er, and the detection sensitivity of single antigen was poorer. Conclusion The structure of all members in the PE family of MTB is analysed, and the recombinant protein pET32a/Rv3388637-731 is expressed and purified success-fully. Seven kinds of proteins in the serum antibody are antigen complementary in the detection. Different antigen is different reaction pattern. A variety of antigens should be jointly detected to improve the sensitivity of antibody de-tection of tuberculosis.
