CAJ | 학술논문

目的克隆和表达梅毒螺旋体Tp0319基因,并对其表达产物进行免疫活性分析.方法挑选并克隆出Tp0319免疫优势区基因,构建原核表达载体;诱导表达并纯化重组蛋白,用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western印迹法鉴定;用纯化的重组蛋白免疫新西兰兔;间接ELISA法检测梅毒螺旋体参考血清及临床标本.结果成功构建原核表达载体pQE32/Tp03 19;高效表达和纯化出一相对分子质量约30 000的重组蛋白.用重组蛋白免疫新西兰兔,能刺激其产生高水平抗体滴度.Western印迹证明其能与梅毒患者血清发生特异性反应,阴性对照菌未见目的表达条带.间接ELISA法检测80份梅毒螺旋体参考血清(阴性、阳性各40份),阳性和阴性结果的符合率均为100%.检测200份临床梅毒血清标本及200份正常人血清,结果与梅毒螺旋体明胶凝集试验相比,灵敏度和特异度分别为92.6%和100%,符合率为96%.结论制备的Tp0319重组蛋白具有良好的免疫活性,为进一步研究其在梅毒血清学诊断中的应用奠定一定的基础.
목적 극륭화표체매독라선체Tp0319기인,병대기표체산물진행면역활성분석.방법 도선병극륭출Tp0319면역우세구기인,구건원핵표체재체;유도표체병순화중조단백,용십이완기류산납-취병희선알응효전영(SDS-PAGE)화Western인적법감정;용순화적중조단백면역신서란토;간접ELISA법검측매독라선체삼고혈청급림상표본.결과 성공구건원핵표체재체pQE32/Tp03 19;고효표체화순화출일상대분자질량약30 000적중조단백.용중조단백면역신서란토,능자격기산생고수평항체적도.Western인적증명기능여매독환자혈청발생특이성반응,음성대조균미견목적 표체조대.간접ELISA법검측80빈매독라선체삼고혈청(음성、양성각40빈),양성화음성결과적부합솔균위100%.검측200빈림상매독혈청표본급200빈정상인혈청,결과여매독라선체명효응집시험상비,령민도화특이도분별위92.6%화100%,부합솔위96%.결론 제비적Tp0319중조단백구유량호적면역활성,위진일보연구기재매독혈청학진단중적응용전정일정적기출.
Objective To clone, express Tp0319 gene from Treponemapallidum (T. pallidum), and to assess the immunocompetence of recombinant protein. Methods The immuno-dominant region of Tp0319gene was chosen by computer analysis, amplified from T. pallidum complete genome by PCR, subcloned into the expression vector pQE32 to construct a recombinant plasmid, pQE32/Tp0319, which was then expressed in E. coli M15. The recombinant protein was purified with Ni-NTA affinity chromatography, and identified by using sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blot. New Zealand rabbits were immunized with the recombinant protein, and the titer of anti-Tp0319 antibodies in sera from immunized rabbits were measured with indirect ELISA. Also, indirect ELISA with the recombinant Tp0319 as coating antigen was performed to detect the anti-Tp0319 antibody in sera from 200 normal human controls and 200 patients with syphilis. Results The prokaryotic expression vector pQE32/Tp0319 was constructed successfully, and the recombinant protein Tp0319 with a molecular weight of about 30 000 was attained. Specific humoral response was elicited by the recombinant protein in New Zealand rabbits and the specific antibody titer was more than 1: 10 240 after immunization for 3 times. Western blot proved that the recombinant protein could specifically react with anti-T. pallidum IgG antibody-positive sera. Indirect ELISA was successfully developed with the recombinant Tp0319, and detected antibodies to T. pallidum in control sera with a sensitivity and specificity of 100% (40/40), respectively. Compared with T. pallidum particle agglutination (TPPA) assay, the sensitivity and specificity of the indirect ELISA were 92.6% and 100%, respectively, in the detection of T. pallidum in sera from patients and controls, and the concordance between the indirect ELISA and TPPA was 96%. Conclusions The prepared recombinant protein shows a satisfactory immunocompetence, which may lay a foundation for its further application in the serodiagnosis of syphilis.