摘要
目的 构建含OrientiatsutsugamushiSxh95 1株 (Ot.Sxh95 1)相对分子质量 (Mr)为 5 6×10 3外膜蛋白基因 (sxh5 6 )的重组质粒pQE30 5 6 ,表达Mr5 6× 10 3蛋白并观察其在ELISA中的应用。方法 IPTG诱导sxh5 6重组质粒 ;观察SDS PAGE和免疫印迹结果 ;经Ni NTA亲和层析纯化后的重组蛋白分别与Ot.Gilliam、Ot.Karp、Ot.Kato感染鼠血清进行ELISA和免疫印迹检测。结果 SDS PAGE和免疫印迹检测显示有一Mr 约为 5 6× 10 3的特异蛋白带 ;ELISA和免疫印迹结果显示该重组蛋白只与Ot.Gilliam感染鼠血清呈阳性反应 ;重组蛋白用作ELISA包被抗原检测小鼠血清抗体IgG ,特异性为 10 0 % ,敏感性 96 .6 7% ;检测人血清抗体IgG的敏感性为 88.0 8% ,特异性 96 .36 %。结论 表达的重组Sxh5 6蛋白具有良好的免疫反应性 ;其作为ELISA包被抗原 ,具有良好的特异性和敏感性。
Objective To explore the expression of the 56kD protein of Orientia tsutsugamushi, which is located on the member surface of Orientia. Methods The gene that encodes the 56kD protein of O.tsutsugamushi Shanxi (sxh56) has been amplified by PCR and cloned into an expression vector pQE30. The 56kD protein of Oriental tsutsugamushi Shanxi was expressed as a fusion protein with the 6×His-binding protein of Escherichia coli. The recombinant protein expressed in Escherichia coli M15 as an inclusion body. Recombinant protein only reacted to polyclonal antiserum to Ot. Gilliam and Ot. Sxh951 in ELISA and immunoblot assay. The recombinant protein was used in an enzyme-linked immunosorbent assay to evaluate the method for the detection of the antibody to O.tsutsugamushi in human and animal sera. Results One hundred and fifty-one positive sera and 412 negative human sera to Ot. Gilliam in IFA were detected with the recombinant protein and the sensitivity was 88.08%, specificity was 96.36%. 30 positive sera and 50 negative animal sera to Ot. Gilliam in IFA were also detected with the recombinant protein and the sensitivity and specificity were 100% and 96.67%. Conclusion The purified sxh56 is a suitable candidate of immunodiagnostic antigen.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2004年第2期155-158,共4页
Chinese Journal of Microbiology and Immunology
