CAJ | 학술논문

目的：克隆Tp0821，Tp0319，Tp0971和Tp0663基因，构建原核表达质粒，进行重组蛋白的表达、纯化并用于检测梅毒病人血清抗体，从而探讨Tp0821，Tp0971，Tp0319和Tp0663重组蛋白在 Tp感染诊断中的作用，丰富梅毒血清学诊断的候选抗原库。方法从Tp库中筛选Tp0821，Tp0319，Tp0971和Tp0663四种膜蛋白，通过生物信息学软件分析挑选优势抗原表位，与 pQE32载体进行连接分别构建 pQE32／Tp0821，pQE32／Tp0319，pQE32／Tp0971和 pQE32／Tp0663原核表达重组体；进行体外克隆表达与纯化，并分别以单一的 Tp0821，Tp0319，Tp0971，Tp0663及四种重组蛋白嵌合抗原（Tp0821-Tp0319-Tp0971-Tp0663）为包被抗原建立间接Tp-ELISA方法，以TRUST和TPPA法为对照，检测2013年1月～2014年6月深圳市宝安区人民医院门诊及住院Tp阴性患者160例和 Tp阳性83例临床标本，评价其在梅毒血清学诊断中的应用。结果成功构建原核表达载体 pQE32／Tp0821，pQE32／Tp0319，pQE32／Tp0971和 pQE32／Tp0663；高效表达和纯化出各自相对分子质量的重组蛋白。建立的间接 Tp-ELISA法检测160例 Tp阴性和 8 3例 Tp阳性临床标本，与TPPA相比，敏感度分别为85.5％（71／83），84.3％（70／83），89.2％（74／83），81.9％（68／83）及95.2％（79／83），特异度均为100％（160／160），符合率为82.6％～95.7％。单一的 Tp0821，Tp0319，Tp0971和 Tp0663重组蛋白建立的 TP-ELISA的阳性检出率（85.5％，84.3％，89.2％及81.9％）与 TPPA法的阳性检出率（100％）比较差异有统计学显著性意义（χ2＝24.5～31.8，P均＜0.01），而四种重组蛋白嵌合抗原建立的 TP-ELISA的阳性检出率与 TPPA法的阳性检出率比较差异有统计学意义（χ2＝7.95，P＜0.05）。结论制备的 Tp0821，Tp0319，Tp0971和 Tp0663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，但以四种重组蛋白嵌合抗原建立的间接 Tp-ELISA法进行Tp血清学诊断，能提高其检测敏感度。目的：剋隆Tp0821，Tp0319，Tp0971和Tp0663基因，構建原覈錶達質粒，進行重組蛋白的錶達、純化併用于檢測梅毒病人血清抗體，從而探討Tp0821，Tp0971，Tp0319和Tp0663重組蛋白在 Tp感染診斷中的作用，豐富梅毒血清學診斷的候選抗原庫。方法從Tp庫中篩選Tp0821，Tp0319，Tp0971和Tp0663四種膜蛋白，通過生物信息學軟件分析挑選優勢抗原錶位，與 pQE32載體進行連接分彆構建 pQE32／Tp0821，pQE32／Tp0319，pQE32／Tp0971和 pQE32／Tp0663原覈錶達重組體；進行體外剋隆錶達與純化，併分彆以單一的 Tp0821，Tp0319，Tp0971，Tp0663及四種重組蛋白嵌閤抗原（Tp0821-Tp0319-Tp0971-Tp0663）為包被抗原建立間接Tp-ELISA方法，以TRUST和TPPA法為對照，檢測2013年1月～2014年6月深圳市寶安區人民醫院門診及住院Tp陰性患者160例和 Tp暘性83例臨床標本，評價其在梅毒血清學診斷中的應用。結果成功構建原覈錶達載體 pQE32／Tp0821，pQE32／Tp0319，pQE32／Tp0971和 pQE32／Tp0663；高效錶達和純化齣各自相對分子質量的重組蛋白。建立的間接 Tp-ELISA法檢測160例 Tp陰性和 8 3例 Tp暘性臨床標本，與TPPA相比，敏感度分彆為85.5％（71／83），84.3％（70／83），89.2％（74／83），81.9％（68／83）及95.2％（79／83），特異度均為100％（160／160），符閤率為82.6％～95.7％。單一的 Tp0821，Tp0319，Tp0971和 Tp0663重組蛋白建立的 TP-ELISA的暘性檢齣率（85.5％，84.3％，89.2％及81.9％）與 TPPA法的暘性檢齣率（100％）比較差異有統計學顯著性意義（χ2＝24.5～31.8，P均＜0.01），而四種重組蛋白嵌閤抗原建立的 TP-ELISA的暘性檢齣率與 TPPA法的暘性檢齣率比較差異有統計學意義（χ2＝7.95，P＜0.05）。結論製備的 Tp0821，Tp0319，Tp0971和 Tp0663重組蛋白具有良好的免疫活性，為進一步研究其在梅毒血清學診斷中的應用奠定一定的基礎，但以四種重組蛋白嵌閤抗原建立的間接 Tp-ELISA法進行Tp血清學診斷，能提高其檢測敏感度。
목적：극륭Tp0821，Tp0319，Tp0971화Tp0663기인，구건원핵표체질립，진행중조단백적표체、순화병용우검측매독병인혈청항체，종이탐토Tp0821，Tp0971，Tp0319화Tp0663중조단백재 Tp감염진단중적작용，봉부매독혈청학진단적후선항원고。방법종Tp고중사선Tp0821，Tp0319，Tp0971화Tp0663사충막단백，통과생물신식학연건분석도선우세항원표위，여 pQE32재체진행련접분별구건 pQE32／Tp0821，pQE32／Tp0319，pQE32／Tp0971화 pQE32／Tp0663원핵표체중조체；진행체외극륭표체여순화，병분별이단일적 Tp0821，Tp0319，Tp0971，Tp0663급사충중조단백감합항원（Tp0821-Tp0319-Tp0971-Tp0663）위포피항원건립간접Tp-ELISA방법，이TRUST화TPPA법위대조，검측2013년1월～2014년6월심수시보안구인민의원문진급주원Tp음성환자160례화 Tp양성83례림상표본，평개기재매독혈청학진단중적응용。결과성공구건원핵표체재체 pQE32／Tp0821，pQE32／Tp0319，pQE32／Tp0971화 pQE32／Tp0663；고효표체화순화출각자상대분자질량적중조단백。건립적간접 Tp-ELISA법검측160례 Tp음성화 8 3례 Tp양성림상표본，여TPPA상비，민감도분별위85.5％（71／83），84.3％（70／83），89.2％（74／83），81.9％（68／83）급95.2％（79／83），특이도균위100％（160／160），부합솔위82.6％～95.7％。단일적 Tp0821，Tp0319，Tp0971화 Tp0663중조단백건립적 TP-ELISA적양성검출솔（85.5％，84.3％，89.2％급81.9％）여 TPPA법적양성검출솔（100％）비교차이유통계학현저성의의（χ2＝24.5～31.8，P균＜0.01），이사충중조단백감합항원건립적 TP-ELISA적양성검출솔여 TPPA법적양성검출솔비교차이유통계학의의（χ2＝7.95，P＜0.05）。결론제비적 Tp0821，Tp0319，Tp0971화 Tp0663중조단백구유량호적면역활성，위진일보연구기재매독혈청학진단중적응용전정일정적기출，단이사충중조단백감합항원건립적간접 Tp-ELISA법진행Tp혈청학진단，능제고기검측민감도。
Objective To clone Tp0821,Tp0319,Tp0971 and Tp0663 gene,construct prokaryotic expression plasmid,the ex-pression of recombinant proteins,purification and used to detect syphilis patients serum antibodies.To explore Tp0821, Tp0971,Tp0319 and Tp0663 recombinant protein in the diagnosis of Tp infection,rich library of candidate antigens syphilis serology diagnosis.Methods From Tp library screening Tp0821,Tp0319,Tp0971 and Tp0663 four kinds of membrane pro-tein,through the analysis of the bioinformatics software selection advantage antigen epitope,connected with pQE32 carrier respectively built pQE32/Tp0821,pQE32/Tp0319,pQE32/Tp0971 and pQE32/Tp0663 prokaryotic expression recombi-nant;In vitro cloning,expression and purification and on single Tp0821,Tp0319,Tp0971,Tp0663 and four kinds of recombi-nant protein chimeric antigen (Tp0821-Tp0319-Tp0971-Tp0663)in order to establish the indirect Tp-envelope antigen ELISA method,based on TRUST and TPPA method comparison,detection collected from January 2013 to June 2014 in Shenzhen Baoan District People’s Hospital outpatient and hospitalization Tp negative patients,160 cases of positive speci-mens of 83 cases of clinical and Tp,and evaluated its application in the syphilis serology diagnosis.Results Successful con-structed of prokaryotic expression vector pQE32/Tp0821,pQE32/Tp0319,pQE32/Tp0971 and pQE32/Tp0663.Efficient expressed and purified their relative molecular mass of recombinant proteins.Seted up Tp-indirect ELISA method to detect 160 cases of Tp negativepositive clinical specimens and 83 cases of Tp,and compared with TPPA,the sensitivity were 85.5% (71/83),84.3% (70/83),89.2% (74/83),81.9% (68/83)and 95.2% (79/83)respectively,specificity was 100%(160/160),and the coincidence rate was 82.6%~95.7%.Single Tp0821,Tp0319,Tp0971 and Tp0663 recombinant protein positive rate of TP-ELISA was established (85.5%,84.3%,89.2% and 81.9%)compared with TPPA method of positive detection rate (100%)had significant difference statistically significant (χ2= 24.5~31.8,P<0.01),and four kinds of re-combinant protein chimeric antigen positive rate of TP-ELISA was established with the TPPA method the positive detection rate of comparative difference was statistically significant (χ2=7.95,P<0.05).Conclusion Preparation Tp0821,Tp0319, Tp0971 and Tp0663 recombinant protein had good immune activity,for the further study of its application in the diagnosis of syphilis serology lay a certain foundation,but in four kinds of recombinant protein chimeric antigen based Tp Tp-indirect ELISA method for serological diagnosis,can improve the detection sensitivity.