本文選題：梅毒螺旋體 + Tp0965��； 參考：《南華大學》2012年碩士論文

【摘要】：目的評價梅毒螺旋體(Treponema pallidum,Tp）重組蛋白rTp0608、rTp0965和rTp1038(TpF1）在梅毒臨床血清學診斷中的意義，為發(fā)現(xiàn)新Tp診斷抗原提供實驗依據(jù)。 方法從Genbank獲取Tp0608和Tp0965基因序列，以Tp Nichols標準株全基因組DNA為模板，PCR擴增其全基因片段；將酶切后的目的片段克隆入原核表達載體pET28a，構建重組載體pET28a-Tp0608和pET28a-Tp0965，經(jīng)酶切和測序鑒定正確后轉化至表達菌E.coli BL21(DE3)中，以IPTG誘導蛋白表達，用SDS-PAGE鑒定蛋白表達形式，Western blot鑒定表達蛋白的免疫反應性；用Ni2+-NTA親和層析柱純化重組蛋白，SDS-PAGE分析純化蛋白的純度；以純化蛋白抗原rTp0608、rTp0965和rTpF1(本室保存）單獨或聯(lián)合包被酶標板，以確診的梅毒陽性血清或陰性血清為一抗，以HRP標記羊抗人IgG為二抗，建立間接ELISA，優(yōu)化最佳的抗原包被濃度、最佳抗原組合以及該組合的最佳抗原包被濃度，最適血清(一抗）稀釋度，篩選出優(yōu)化后最佳抗原組合的ELISA并檢測其重復性；用已優(yōu)化的rTpF1-rTp0965-ELISA檢測確診的梅毒陽性和陰性血清，統(tǒng)計學分析該方法的敏感性、特異性和一致性；進一步隨機檢測各期TPPA陽性梅毒血清、TPPA陰性的易發(fā)生交叉反應人群(系統(tǒng)性紅斑狼瘡、類風濕關節(jié)炎、結核病）血清及正常健康人對照血清，同時與TPPA、國產(chǎn)Tp-ELISA試劑盒進行平行比較，初步評價rTpF1-rTp0965-ELISA在梅毒血清學診斷中的應用價值。 結果 (1) PCR分別擴增出一大小約888bp和960bp的目的片段(含引物），重組質(zhì)粒經(jīng)雙酶切、測序鑒定與GenBank上公布的序列基本一致，表明插入的目的基因為Tp0608和Tp0965。 (2)重組表達載體pET28a-Tp0608和pET28a-Tp0965在宿主菌E.coliBL21(DE3)被IPTG誘導分別表達出相對分子量約為38kDa和43kDa的目的蛋白；表達形式鑒定顯示，Tp0608在菌體細胞中以包涵體形式存在，而Tp0965則以可溶性蛋白的形式存在。 (3)經(jīng)Ni2+-NTA親和層析柱純化后，rTp0608和rTp0965純度均在95%以上；Western blot結果顯示均與梅毒陽性血清有良好的免疫反應性，而與正常人的血清不反應。 (4) ELISA優(yōu)化結果顯示，單抗原包被以TpF1(4μg/Ml）檢測A值比(P/N）最高，Tp0608(2μg/Ml）最低；多抗原包被以TpF1(4μg/Ml）+Tp0965(4μg/Ml）檢測A值比(P/N）最高；最適血清的反應稀釋度為1:200。最佳抗原組合ELISA的批內(nèi)和批間重復性試驗顯示，該法的平均變異系數(shù)(CV）值均為2.6%(10%）。 (5)用已優(yōu)化的rTpF1-rTp0965-ELISA方法檢測31例確診梅毒陽性和19例陰性血清，其敏感性和特異性分別為96.7%和100%，總符合率為98.0%；對93份臨床血清標本隨機進行檢測，與TPPA法比較，建立的ELISA法的總靈敏度為96.8%，低于TPPA法(P＜0.05)，特異度為100%，，兩種方法的總符合率為97.6%；建立的ELISA法與國產(chǎn)Tp-ELISA試劑盒靈敏度均無顯著性差異，特異度高于國產(chǎn)Tp-ELISA試劑盒(P＜0.05)。 結論 (1)成功表達重組蛋白Tp0608和Tp0965，兩蛋白有良好的免疫反應性。 (2) Tp0608可能不宜作為診斷抗原，TpF1和Tp0965可望成為候選診斷抗原；建立的rTpF1-rTp0965-ELISA具有高度特異性、較好的敏感性和重復性，但其臨床應用有待進一步評價和驗證。
[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.

【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R759.1;R446.5

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