梅毒螺旋体候选诊断抗原的表达及临床初步应用

发布时间：2018-04-23 20:17

本文选题：梅毒螺旋体 + Tp0965　；参考：《南华大学》2012年硕士论文

【摘要】：目的评价梅毒螺旋体(Treponema pallidum,Tp）重组蛋白rTp0608、rTp0965和rTp1038(TpF1）在梅毒临床血清学诊断中的意义，为发现新Tp诊断抗原提供实验依据。方法从Genbank获取Tp0608和Tp0965基因序列，以Tp Nichols标准株全基因组DNA为模板，PCR扩增其全基因片段；将酶切后的目的片段克隆入原核表达载体pET28a，构建重组载体pET28a-Tp0608和pET28a-Tp0965，经酶切和测序鉴定正确后转化至表达菌E.coli BL21(DE3)中，以IPTG诱导蛋白表达，用SDS-PAGE鉴定蛋白表达形式，Western blot鉴定表达蛋白的免疫反应性；用Ni2+-NTA亲和层析柱纯化重组蛋白，SDS-PAGE分析纯化蛋白的纯度；以纯化蛋白抗原rTp0608、rTp0965和rTpF1(本室保存）单独或联合包被酶标板，以确诊的梅毒阳性血清或阴性血清为一抗，以HRP标记羊抗人IgG为二抗，建立间接ELISA，优化最佳的抗原包被浓度、最佳抗原组合以及该组合的最佳抗原包被浓度，最适血清(一抗）稀释度，筛选出优化后最佳抗原组合的ELISA并检测其重复性；用已优化的rTpF1-rTp0965-ELISA检测确诊的梅毒阳性和阴性血清，统计学分析该方法的敏感性、特异性和一致性；进一步随机检测各期TPPA阳性梅毒血清、TPPA阴性的易发生交叉反应人群(系统性红斑狼疮、类风湿关节炎、结核病）血清及正常健康人对照血清，同时与TPPA、国产Tp-ELISA试剂盒进行平行比较，初步评价rTpF1-rTp0965-ELISA在梅毒血清学诊断中的应用价值。结果 (1) PCR分别扩增出一大小约888bp和960bp的目的片段(含引物），重组质粒经双酶切、测序鉴定与GenBank上公布的序列基本一致，表明插入的目的基因为Tp0608和Tp0965。 (2)重组表达载体pET28a-Tp0608和pET28a-Tp0965在宿主菌E.coliBL21(DE3)被IPTG诱导分别表达出相对分子量约为38kDa和43kDa的目的蛋白；表达形式鉴定显示，Tp0608在菌体细胞中以包涵体形式存在，而Tp0965则以可溶性蛋白的形式存在。 (3)经Ni2+-NTA亲和层析柱纯化后，rTp0608和rTp0965纯度均在95%以上；Western blot结果显示均与梅毒阳性血清有良好的免疫反应性，而与正常人的血清不反应。 (4) ELISA优化结果显示，单抗原包被以TpF1(4μg/Ml）检测A值比(P/N）最高，Tp0608(2μg/Ml）最低；多抗原包被以TpF1(4μg/Ml）+Tp0965(4μg/Ml）检测A值比(P/N）最高；最适血清的反应稀释度为1:200。最佳抗原组合ELISA的批内和批间重复性试验显示，该法的平均变异系数(CV）值均为2.6%(10%）。 (5)用已优化的rTpF1-rTp0965-ELISA方法检测31例确诊梅毒阳性和19例阴性血清，其敏感性和特异性分别为96.7%和100%，总符合率为98.0%；对93份临床血清标本随机进行检测，与TPPA法比较，建立的ELISA法的总灵敏度为96.8%，低于TPPA法(P＜0.05)，特异度为100%，，两种方法的总符合率为97.6%；建立的ELISA法与国产Tp-ELISA试剂盒灵敏度均无显著性差异，特异度高于国产Tp-ELISA试剂盒(P＜0.05)。结论 (1)成功表达重组蛋白Tp0608和Tp0965，两蛋白有良好的免疫反应性。 (2) Tp0608可能不宜作为诊断抗原，TpF1和Tp0965可望成为候选诊断抗原；建立的rTpF1-rTp0965-ELISA具有高度特异性、较好的敏感性和重复性，但其临床应用有待进一步评价和验证。
[Abstract]:Objective to evaluate the significance of Treponema pallidum (Tp) recombinant protein rTp0608, rTp0965 and rTp1038 (TpF1) in the clinical serological diagnosis of syphilis, and to provide an experimental basis for the discovery of the new Tp diagnostic antigen.
Methods the Tp0608 and Tp0965 gene sequences were obtained from Genbank, and the whole genome DNA of Tp Nichols standard strain was used as template, and the whole gene fragment was amplified by PCR, and the target fragment was cloned into the prokaryotic expression vector pET28a, and the recombinant vector pET28a-Tp0608 and pET28a-Tp0965 were constructed, and then converted to the E.coli BL21 (E.coli BL21) by enzyme digestion and sequencing. DE3), the protein expression was induced by IPTG, the protein expression was identified by SDS-PAGE, the immunoreactivity of the expressed protein was identified by Western blot, the recombinant protein was purified by Ni2+-NTA affinity chromatography column, the purity of the protein was purified by SDS-PAGE, and the purified protein antigen rTp0608, rTp0965 and rTpF1 (preserved in this chamber) were separately or jointly coated with the enzyme plate. The positive sera or negative sera of the syphilis were one resistance, and the HRP labeled Sheep anti human IgG was two, and the indirect ELISA was established to optimize the best antigen inclusion concentration, the best antigen combination and the optimum antigen concentration, the optimum serum (one anti) dilution degree, the optimum antigen combination ELISA was screened and its reproducibility was detected. The positive and negative sera were detected by the optimized rTpF1-rTp0965-ELISA, and the sensitivity, specificity and consistency of the method were statistically analyzed. The serum and normal health of TPPA positive syphilis, TPPA negative cross reacting population (systemic lupus erythematosus, rheumatoid arthritis, tuberculosis) were further randomized. Parallel comparison of human serum with TPPA and domestic Tp-ELISA kit was performed to evaluate the value of rTpF1-rTp0965-ELISA in serologic diagnosis of syphilis.
Result
(1) the target fragment (including primers) of 888bp and 960bp was amplified by PCR, and the recombinant plasmid was cut by double enzyme. The sequencing identification was basically consistent with the sequence published on GenBank, indicating that the target gene inserted was Tp0608 and Tp0965..
(2) the recombinant expression vector pET28a-Tp0608 and pET28a-Tp0965 were induced by IPTG in the host bacteria E.coliBL21 (DE3) to express the relative molecular weight of about 38kDa and 43kDa, respectively. The expression identification showed that Tp0608 existed in the form of inclusion body in the mycelial cells and Tp0965 in the form of soluble protein.
(3) the purity of rTp0608 and rTp0965 was above 95% after purification by Ni2+-NTA affinity chromatography column, and the results of Western blot showed that all of them had good immunoreactivity with the positive sera of syphilis, but did not react with the normal human serum.
(4) ELISA optimization results show that TpF1 (P/N) is the highest and Tp0608 (2 g/Ml) is the lowest in the primary package of McAb, and Tp0608 (2 mu g/Ml) is the lowest. The maximum of the polyclonal package is TpF1 (4 mu g/Ml) +Tp0965 (4) +Tp0965 (4 mu g/Ml). The coefficient of variation (CV) was 2.6% (10%).
(5) 31 cases of confirmed syphilis positive and 19 negative sera were detected by the optimized rTpF1-rTp0965-ELISA method. The sensitivity and specificity were 96.7% and 100% respectively, and the total coincidence rate was 98%. 93 clinical serum specimens were randomly tested. Compared with the TPPA method, the total sensitivity of the established ELISA method was 96.8%, lower than the TPPA method (P < 0.05), specificity. For 100%, the total coincidence rate of the two methods was 97.6%, and the sensitivity of the established ELISA method and the domestic Tp-ELISA kit had no significant difference, and the specificity was higher than that of the domestic Tp-ELISA Kit (P < 0.05).
conclusion
(1) the recombinant protein Tp0608 and Tp0965 were successfully expressed, and the two protein had good immunoreactivity.
(2) Tp0608 may not be a diagnostic antigen, and TpF1 and Tp0965 may be expected to be candidate diagnostic antigens; the established rTpF1-rTp0965-ELISA has high specificity, good sensitivity and reproducibility, but its clinical application needs further evaluation and verification.

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R759.1;R446.5

【参考文献】