中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2011年
2期
119-123
,共5页
杨珺%沈琳%张小贤%孙琦
楊珺%瀋琳%張小賢%孫琦
양군%침림%장소현%손기
梅毒螺旋体%Tp0136%活性肽段%可溶性表达%免疫活性
梅毒螺鏇體%Tp0136%活性肽段%可溶性錶達%免疫活性
매독라선체%Tp0136%활성태단%가용성표체%면역활성
Treponema pallidum%Tp0136%Selective fragment%Soluble expression%Immunity activity
目的 筛选梅毒螺旋体特异性抗原Tp0136的活性肽段,可溶性表达和纯化该肽段,并鉴定其免疫活性,探索Tp0136活性肽段在早期梅毒诊断中的价值.方法 通过生物信息学方法对Tp0136亲水性、B细胞表位和二级结构等进行分析,筛选出Tp0136活性肽段(Tp0136B)替代全蛋白.将Tp0136B基因插入到pET22b(+)上,在E.coli BL21中表达.镍离子亲和色谱纯化表达产物,Western blot检测其免疫反应性,免疫日本大耳白兔评价其免疫原性,免疫双扩检测其效价,以重组Tp0136B蛋白为包被抗原的间接ELISA检测早期梅毒血清抗体.结果 重组工程菌可溶性表达相对分子质量约为28×103的rTp0136B,表达率为21%,制备得到纯度大于98%的rTp0136B.纯化的rTp0136B能诱导大耳白兔产生特异性免疫应答,免疫双扩测得其效价为1:16.Western blot检测重组蛋白能与兔抗Tp0136多克隆抗体发生特异性反应.间接ELISA检测正常人血清均为阴性,而早期梅毒血清抗体的阳性率为85.5%.结论 重组表达的Tp0136活性肽段具有良好的免疫活性,预示其在早期梅毒血清学诊断中具有良好的前景.
目的 篩選梅毒螺鏇體特異性抗原Tp0136的活性肽段,可溶性錶達和純化該肽段,併鑒定其免疫活性,探索Tp0136活性肽段在早期梅毒診斷中的價值.方法 通過生物信息學方法對Tp0136親水性、B細胞錶位和二級結構等進行分析,篩選齣Tp0136活性肽段(Tp0136B)替代全蛋白.將Tp0136B基因插入到pET22b(+)上,在E.coli BL21中錶達.鎳離子親和色譜純化錶達產物,Western blot檢測其免疫反應性,免疫日本大耳白兔評價其免疫原性,免疫雙擴檢測其效價,以重組Tp0136B蛋白為包被抗原的間接ELISA檢測早期梅毒血清抗體.結果 重組工程菌可溶性錶達相對分子質量約為28×103的rTp0136B,錶達率為21%,製備得到純度大于98%的rTp0136B.純化的rTp0136B能誘導大耳白兔產生特異性免疫應答,免疫雙擴測得其效價為1:16.Western blot檢測重組蛋白能與兔抗Tp0136多剋隆抗體髮生特異性反應.間接ELISA檢測正常人血清均為陰性,而早期梅毒血清抗體的暘性率為85.5%.結論 重組錶達的Tp0136活性肽段具有良好的免疫活性,預示其在早期梅毒血清學診斷中具有良好的前景.
목적 사선매독라선체특이성항원Tp0136적활성태단,가용성표체화순화해태단,병감정기면역활성,탐색Tp0136활성태단재조기매독진단중적개치.방법 통과생물신식학방법대Tp0136친수성、B세포표위화이급결구등진행분석,사선출Tp0136활성태단(Tp0136B)체대전단백.장Tp0136B기인삽입도pET22b(+)상,재E.coli BL21중표체.얼리자친화색보순화표체산물,Western blot검측기면역반응성,면역일본대이백토평개기면역원성,면역쌍확검측기효개,이중조Tp0136B단백위포피항원적간접ELISA검측조기매독혈청항체.결과 중조공정균가용성표체상대분자질량약위28×103적rTp0136B,표체솔위21%,제비득도순도대우98%적rTp0136B.순화적rTp0136B능유도대이백토산생특이성면역응답,면역쌍확측득기효개위1:16.Western blot검측중조단백능여토항Tp0136다극륭항체발생특이성반응.간접ELISA검측정상인혈청균위음성,이조기매독혈청항체적양성솔위85.5%.결론 중조표체적Tp0136활성태단구유량호적면역활성,예시기재조기매독혈청학진단중구유량호적전경.
Objective To express and purify recombinant Tp0136 epitope fragment, and study the immunity activity. Methods The Tp0136 selective fragment(Tp0136B) gene was devised by the surface property analysis, solvent-accessible suface calculateions, secondary structure function region analysis, and was inserted between the sites of Nde Ⅰ and Not Ⅰ in pET22b ( + ) . The recombinant plasmid was expressed in E. coli BI21. After nickel ion metal affinity chromatography, the antigenic and immune reactivity of rTp0136B was confirmed. Then indirect ELISA with the rTp0136B as coating antigen was performed to detect the anti-Tp0136 antibody in sera from 100 normal human controls and 131 primary syphilis patients. Results The rTp0136B was soluble expressed with a molecular weight of about 28 000 and was obtained with a purity of ＞98% by chromatography. Western blot proved that the rTp0136B could specifically react with anti-Tp0136 polyclonal antibody. Specific humoral response was elicited by the recombinant protein in Japan negative. The positive detection rate in sera from primary syphilis patients was 85.5%. Conclusion This result suggested that the recombinant Tp0136 epitope fragments have a satisfactory immunocompetence,which may have applications in the serodiagnosis of primary syphilis.
