[1]房笃智,阳 建,杨荣志,等.梅毒螺旋体四种膜蛋白 克隆重组表达和ELISA法建立的应用研究[J].现代检验医学杂志,2015,30(01):75-77.[doi:10.3969/j.issn.1671-7414.2015.01.020]
　FANG Du-zhi,YANG Jian,YANG Rong-zhi,et al.Cloning Recombinant Expression of Treponema Pallidum Four Kinds of Membrane Protein and Application of Establishing ELISA Method[J].Journal of Modern Laboratory Medicine,2015,30(01):75-77.[doi:10.3969/j.issn.1671-7414.2015.01.020]
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梅毒螺旋体四种膜蛋白 克隆重组表达和ELISA法建立的应用研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第30卷
期数:
2015年01期
页码:
75-77
栏目:
论著
出版日期:
2015-01-30

文章信息/Info

Title:
Cloning Recombinant Expression of Treponema Pallidum Four Kinds of Membrane Protein and Application of Establishing ELISA Method
文章编号:
1671-7414(2015)01-075-04
作者:
房笃智1; 阳 建2; 杨荣志2; 唐 诚2
1.深圳市宝安区人民医院检验科,广东深圳 518109;
2.深圳市宝安区沙井人民医院检验科,广东深圳 518104
Author(s):
FANG Du-zhi1; YANG Jian2; YANG Rong-zhi2; TANGCheng2
1.Department of Clinical Laboratory,the Baoan District People's Hospital of Shenzhen,Guangdong Shenzhen 518109,China;
2.Department of Clinical Laboratory, the Shajing People's Hospital of Baoan District,Guangdong Senzhen 518104,China
关键词:
梅毒螺旋体;  Tp0821;  Tp0319;  Tp0971;  Tp0663;  酶联免疫吸附试验;  血清学试验
分类号:
R377.1; R446.61
DOI:
10.3969/j.issn.1671-7414.2015.01.020
文献标志码:
A
摘要:
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83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。
Abstract:
Objective To clone Tp0821,Tp0319,Tp0971 and Tp0663 gene,construct prokaryotic expression plasmid,the expression 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 protein,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 recombinant; In vitro cloning,expression and purification and on single Tp0821,Tp0319,Tp0971,Tp0663 and four kinds of recombinantprotein chimeric antigen(Tp0821-Tp0319-Tp0971-Tp0663)in order to establishthe indirect Tp-envelope antigen ELISA method,based on TRUST and TPPA method comparison,detection collected from January 2013 to June 2014 in Shenzhen BaoanDistrict People's Hospital outpatient and hospitalization Tp negative patients,160 cases of positive specimens of 83 cases of clinical and Tp,and evaluatedits application in the syphilis serology diagnosis.Results Successful constructed 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 methodto 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 was100%(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 recombinant protein chimeric antigen positive rate of TP-ELISA was established with the TPPA method the positivedetection 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.

参考文献/References:

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备注/Memo

备注/Memo:
基金项目:深圳市宝安区科技局资助,项目编号:2013151。
作者简介:房笃智(1981-),男,本科,主管技师,主要从事临床检验与免疫血库工作,E-mail:curious1997@163.com。
更新日期/Last Update: 2015-01-30