中国免疫学杂志
中國免疫學雜誌
중국면역학잡지
CHINESE JOURNAL OF IMMUNOLOGY
2014年
6期
784-788,796
,共6页
穆柳青%李岱容%张春燕%樊宇%何永林%杨春
穆柳青%李岱容%張春燕%樊宇%何永林%楊春
목류청%리대용%장춘연%번우%하영림%양춘
结核分枝杆菌%rClpC2%兔抗血清
結覈分枝桿菌%rClpC2%兔抗血清
결핵분지간균%rClpC2%토항혈청
Mycobacterium-tuberculosis%rClpC2%Antiserum
目的：分别探索结核分枝杆菌（ Mycobacterium tuberculosis ，MTB） ClpC2蛋白和兔抗ClpC2多克隆抗体作为肺结核检测抗原或抗体的可行性。方法：诱导表达重组蛋白ClpC2（rClpC2）；SDS-PAGE与Western blot 鉴定rClpC2后利用亲和层析法进行纯化；纯化后的重组蛋白免疫新西兰大白兔，Western blot检测兔抗血清的特异性；双向免疫扩散法与酶联免疫吸附法（ ELISA）测定兔抗血清效价；rClpC2通过间接ELISA进行抗原性的初步检测，兔抗ClpC2多克隆抗体通过间接ELISA检测肺结核病人血清中ClpC2抗原。结果：rClpC2经SDS-PAGE分析，在相对分子质量46 kD处可见特异性条带；Western blot分析rClpC2可与MTB H37 Rv株感染的小鼠血清发生抗原抗体反应；经Bandscan分析rClpC2约占菌体总蛋白的58.7％，纯化后rClpC2的纯度为88.5％；Western blot验证兔抗血清可与卡介苗（Bacillus calmette-guerin，BCG）的ClpC2蛋白发生抗原抗体反应；双向免疫扩散法测定兔抗血清效价为1∶32，ELISA法测定兔抗血清效价为1∶320000；rClpC2在检测肺结核病人中的检出率为46％，检测敏感性为46％，特异性为90％；兔抗ClpC2多克隆抗体在检测肺结核病人中的检出率为40％，检测的敏感性为40％，特异性为90％。结论：成功纯化结核分枝杆菌ClpC2蛋白，制备特异性兔抗ClpC2多克隆抗体，为进一步研究ClpC2蛋白的生物功能、ClpC2作为诊断靶标、药靶候选蛋白的可行性及兔抗ClpC2多克隆抗体生物学功能提供实验基础。
目的：分彆探索結覈分枝桿菌（ Mycobacterium tuberculosis ，MTB） ClpC2蛋白和兔抗ClpC2多剋隆抗體作為肺結覈檢測抗原或抗體的可行性。方法：誘導錶達重組蛋白ClpC2（rClpC2）；SDS-PAGE與Western blot 鑒定rClpC2後利用親和層析法進行純化；純化後的重組蛋白免疫新西蘭大白兔，Western blot檢測兔抗血清的特異性；雙嚮免疫擴散法與酶聯免疫吸附法（ ELISA）測定兔抗血清效價；rClpC2通過間接ELISA進行抗原性的初步檢測，兔抗ClpC2多剋隆抗體通過間接ELISA檢測肺結覈病人血清中ClpC2抗原。結果：rClpC2經SDS-PAGE分析，在相對分子質量46 kD處可見特異性條帶；Western blot分析rClpC2可與MTB H37 Rv株感染的小鼠血清髮生抗原抗體反應；經Bandscan分析rClpC2約佔菌體總蛋白的58.7％，純化後rClpC2的純度為88.5％；Western blot驗證兔抗血清可與卡介苗（Bacillus calmette-guerin，BCG）的ClpC2蛋白髮生抗原抗體反應；雙嚮免疫擴散法測定兔抗血清效價為1∶32，ELISA法測定兔抗血清效價為1∶320000；rClpC2在檢測肺結覈病人中的檢齣率為46％，檢測敏感性為46％，特異性為90％；兔抗ClpC2多剋隆抗體在檢測肺結覈病人中的檢齣率為40％，檢測的敏感性為40％，特異性為90％。結論：成功純化結覈分枝桿菌ClpC2蛋白，製備特異性兔抗ClpC2多剋隆抗體，為進一步研究ClpC2蛋白的生物功能、ClpC2作為診斷靶標、藥靶候選蛋白的可行性及兔抗ClpC2多剋隆抗體生物學功能提供實驗基礎。
목적：분별탐색결핵분지간균（ Mycobacterium tuberculosis ，MTB） ClpC2단백화토항ClpC2다극륭항체작위폐결핵검측항원혹항체적가행성。방법：유도표체중조단백ClpC2（rClpC2）；SDS-PAGE여Western blot 감정rClpC2후이용친화층석법진행순화；순화후적중조단백면역신서란대백토，Western blot검측토항혈청적특이성；쌍향면역확산법여매련면역흡부법（ ELISA）측정토항혈청효개；rClpC2통과간접ELISA진행항원성적초보검측，토항ClpC2다극륭항체통과간접ELISA검측폐결핵병인혈청중ClpC2항원。결과：rClpC2경SDS-PAGE분석，재상대분자질량46 kD처가견특이성조대；Western blot분석rClpC2가여MTB H37 Rv주감염적소서혈청발생항원항체반응；경Bandscan분석rClpC2약점균체총단백적58.7％，순화후rClpC2적순도위88.5％；Western blot험증토항혈청가여잡개묘（Bacillus calmette-guerin，BCG）적ClpC2단백발생항원항체반응；쌍향면역확산법측정토항혈청효개위1∶32，ELISA법측정토항혈청효개위1∶320000；rClpC2재검측폐결핵병인중적검출솔위46％，검측민감성위46％，특이성위90％；토항ClpC2다극륭항체재검측폐결핵병인중적검출솔위40％，검측적민감성위40％，특이성위90％。결론：성공순화결핵분지간균ClpC2단백，제비특이성토항ClpC2다극륭항체，위진일보연구ClpC2단백적생물공능、ClpC2작위진단파표、약파후선단백적가행성급토항ClpC2다극륭항체생물학공능제공실험기출。
Objective:To investigate the antigenicity of ClpC 2 and the feasibility of polyclonal antibodies of ClpC 2 as detected antibody.Methods:rClpC2 was induced with IPTG.The rClpC2 was identified by SDS-PAGE and Western blot ,and purified by affinity chromatography ,with which rabbit were immunized and the specificity of rabbit antiserum was detected by Western blot , the titer of rabbit antiserum against ClpC2 was detected by double immunodiffusion and indirect enzyme-linked immunosorbent assay (ELISA).The antigenicity of the rClpC2 was detected by ELISA.The polyclonal antibodies of ClpC 2 were prepared to detect the ClpC 2 in clinical serum of TB patients by ELISA.Results:SDS-PAGE showed specific protein band with a relative molecular mass of 46 kD.The rClpC2 could bind with the antibody in the blood serum of the mouse immuned by MTB.By Bandscan analysis rClpC 2 accounted for about 58.7%of the total bacteria protein ,the purity of rClpC2 was 88.5% after purification.The ClpC2 of BCG could bind with the rabbit antiserum.The titer of antiserum were 1∶32 and 1∶320 000 by double immunodiffusion and ELISA detected respectively.ELISA results showed that clinical serum positive rate of rClpC 2 antigen was 46%in TB patients,the sensitivity of this protein was 46%,and the spe-cificity of this protein was 90%.ELISA results showed that the sensitivity of rabbit antiserum against ClpC 2 was 40%, and the specificity was 90%.Conclusion: Successfully expressed and purified rClpC 2 and high titer polyclonal antibody were successfully prepared,and these results will provide basements for further study on the biological functions of ClpC 2 and its candidate potentiality as serological diagnosis and drug-target and biological functions of antiserum against ClpC 2.
