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
6期
544-548
,共5页
薛冠华%孙红妹%赵汉青%王洛平%冯燕玲%李少丽
薛冠華%孫紅妹%趙漢青%王洛平%馮燕玲%李少麗
설관화%손홍매%조한청%왕락평%풍연령%리소려
肺炎支原体%黏附蛋白%抗原%表达纯化%免疫原性
肺炎支原體%黏附蛋白%抗原%錶達純化%免疫原性
폐염지원체%점부단백%항원%표체순화%면역원성
Mycoplasma pneumoniae%Adhesin protein%Antigen%Expression%Immunogenicity
目的 探索肺炎支原体(Mycoplasma pneumoniae,Mp)P1蛋白作为抗原成分在临床血清抗体检测中的应用.方法 通过生物信息学分析P1基因主要抗原决定簇,选择其分值较高的部位进行原核表达、纯化,并进行Western blot鉴定,用纯化后的蛋白免疫小鼠,评价其免疫原性,同时用其作为抗原,采用ELISA方法检测肺炎支原体患儿血清抗体并与全菌体抗原进行比较.结果 成功构建了pET-28C-P1-534原核表达载体,经表达、纯化后获得了相对分子质量为26×103的融合蛋白;Western blot检测其能与Mp阳性血清发生特异性反应;利用纯化的重组蛋白免疫小鼠,能诱导小鼠产生特异性免疫应答,抗体滴度可达 1∶2000;用此蛋白作为抗原成分检测患儿血清,其敏感性和特异性分别为85.00%及97.67%.结论 重组表达的P1-534蛋白具有良好的免疫原性,作为新的抗原成分其敏感性和特异性优于全菌体抗原,为肺炎支原体抗原成分及其蛋白保护作用的研究奠定了基础.
目的 探索肺炎支原體(Mycoplasma pneumoniae,Mp)P1蛋白作為抗原成分在臨床血清抗體檢測中的應用.方法 通過生物信息學分析P1基因主要抗原決定簇,選擇其分值較高的部位進行原覈錶達、純化,併進行Western blot鑒定,用純化後的蛋白免疫小鼠,評價其免疫原性,同時用其作為抗原,採用ELISA方法檢測肺炎支原體患兒血清抗體併與全菌體抗原進行比較.結果 成功構建瞭pET-28C-P1-534原覈錶達載體,經錶達、純化後穫得瞭相對分子質量為26×103的融閤蛋白;Western blot檢測其能與Mp暘性血清髮生特異性反應;利用純化的重組蛋白免疫小鼠,能誘導小鼠產生特異性免疫應答,抗體滴度可達 1∶2000;用此蛋白作為抗原成分檢測患兒血清,其敏感性和特異性分彆為85.00%及97.67%.結論 重組錶達的P1-534蛋白具有良好的免疫原性,作為新的抗原成分其敏感性和特異性優于全菌體抗原,為肺炎支原體抗原成分及其蛋白保護作用的研究奠定瞭基礎.
목적 탐색폐염지원체(Mycoplasma pneumoniae,Mp)P1단백작위항원성분재림상혈청항체검측중적응용.방법 통과생물신식학분석P1기인주요항원결정족,선택기분치교고적부위진행원핵표체、순화,병진행Western blot감정,용순화후적단백면역소서,평개기면역원성,동시용기작위항원,채용ELISA방법검측폐염지원체환인혈청항체병여전균체항원진행비교.결과 성공구건료pET-28C-P1-534원핵표체재체,경표체、순화후획득료상대분자질량위26×103적융합단백;Western blot검측기능여Mp양성혈청발생특이성반응;이용순화적중조단백면역소서,능유도소서산생특이성면역응답,항체적도가체 1∶2000;용차단백작위항원성분검측환인혈청,기민감성화특이성분별위85.00%급97.67%.결론 중조표체적P1-534단백구유량호적면역원성,작위신적항원성분기민감성화특이성우우전균체항원,위폐염지원체항원성분급기단백보호작용적연구전정료기출.
Objective To study the application of P1 adhesin protein epitopes in diagnosis of Mycoplasma pneumoniae(Mp) infected patient. Methods The major epitope(P1-534) of P1 adhesin protein were predicted by ProPred and ANTIGENIC according to its primary structure. The high value fragment was cloned into a constructed recombinant vector. The gene was induced to express fusion protein in E. coli host strain BL21(DE3) and the fusion protein was identified by Western blot. BALB/c mice were immunized with purified protein to test its immunogenicity. Then the purified protein was used as antigen to test the serum of Mp infected patient by ELISA, and compared with the Mp whole cell antigen. Results The P1-534 protein was successfully expressed and purified. ELISA data showed that P1-534 protein could elicit high levels of IgG in immunized mice, the sensitivity and specificity of P1-534 were determined to be 85.00% and 97.67%, while the Mp whole cell antigen were 72.50% and 74.42%. Conclusion The results conformed that the recombinant epitope has certain immunogenicity,and its sensitivity and specificity are better than Mp whole cell antigen. P1-534 protein can be used as an antigen for immunodiagnosis of Mp infection.
