摘要
目的探讨分泌型蛋白在沙眼衣原体(Ct)活动性感染中的诊断价值。方法通过基因工程方法筛选目的分泌型蛋白,构建重组质粒,转化至表达宿主菌中进行诱导表达,利用Western-Blot分析和鉴定表达产物。以纯化的重组蛋白作为包被抗原,建立其血清学诊断的间接ELISA法,并用已有的IgG试剂盒与其共同检测Ct感染血清标本,评价二者敏感度与特异性,分析该抗血清在临床标本抗原检测中的应用价值。结果经1.5%琼脂糖凝胶电泳,目的基因Pgp3在对应的位置上(大小约564 bp)可见清晰的条带,与预期片段一致。pET30a/Pgp3经表达引物扩增,在对应的位置上(大小约792 bp)可见清晰的条带,经NdeⅠ和HindⅢ双酶切,得到大小约为792 bp的清晰条带。pET30a/Pgp3表达蛋白的诱导剂IPTG温度为10℃,浓度为0.6 mmol/L出现相应的蛋白条带最粗。阴性血清中无重组蛋白的表达,阳性血清有一条反应较弱的明显条带,抗HIS标签有一个明显的清晰条带。阳性标本中,重组蛋白Pgp3的敏感度为86.21%(50/58),特异度为93.83%(152/162),与CtIgG试剂盒符合率为91.82%[(50+152)/220],两种检测结果比较,差异无统计学意义(χ^(2)=0.056,P=0.814)。结论纯化的Ct重组蛋白Pgp3具有较好的免疫原性,以Pgp3蛋白建立的间接ELISA法的具有较好的特异性和敏感度,且与商品CtIgG ELISA试剂盒检测结果符合率高,可能有望作为Ct活动期感染的血清学诊断的候选抗原。
Objective To investigate the diagnostic value of secreted protein in active infection of Chlamydia trachomatis(Ct). Methods The target secreted protein was screened by genetic engineering method,and the recombinant plasmid was constructed,transformed into the expression host bacteria for induced expression,and the expression product was analyzed and identified by Western-Blot. Using the purified recombinant protein as the coating antigen, an indirect ELISA method for serological diagnosis was established,and the existing IgG kit was used to detect Ct-infected serum samples together to evaluate the sensitivity and specificity of the two,and to analyze the clinical application of the antiserum. Result After 1.5% agarose gel electrophoresis,the target gene Pgp3 showed a clear band at the corresponding position(about 564 bp in size),which was consistent with the expected fragment. pET30a/Pgp3 was amplified by expression primers,and a clear band was visible at the corresponding position(about 792 bp in size). After double digestion with Nde Ⅰ and Hind Ⅲ,a clear band with a size of about 792 bp was obtained. The temperature of IPTG,the inducer of p ET30 a/Pgp3 expression protein,was 10℃ and the concentration was 0.6 mmol/L,and the corresponding protein band was the thickest. There was no recombinant protein expression in the negative serum,a clear band with weak reaction in the positive serum,and an obvious clear band in the anti-HIS tag. In the positive samples,the sensitivity of recombinant protein Pgp3 was 86.21%(50/58),the specificity was 93.83%(152/162),and the coincidence rate with the Ct Ig G kit was 91.82%[(50+152)/220],There was no significant difference between the two test results(χ^(2)=0.056,P=0.814). Conclusion The purified Ct recombinant protein Pgp3 has good immunogenicity,and the indirect ELISA method established with Pgp3 protein has good specificity and sensitivity,and has a high coincidence rate with the detection results of commercial CtIgG ELISA kits,which may be expected as a candidate anti
作者
袁红霞
龙歆孜
陈虹亮
周安文
蔡友香
罗丽裴
YUAN Hongxia;LONG Xinzi;CHEN Hongliang;ZHOU Anwen;CAI Youxiang;LUO Lipei(Department of Clinical Laboratory,the First People's Hospital of Chenzhou,Chenzhou,Hunan,China,423000;Department of Laboratory,Zhuzhou Center for Disease Control and prevention,Zhuzhou,Hunan,China,412000;Department of Burn Injury,the First People's Hospital of Chenzhou,Chenzhou,Hunan,China,423000)
出处
《分子诊断与治疗杂志》
2022年第7期1089-1093,共5页
Journal of Molecular Diagnostics and Therapy
基金
湖南省郴州市科技局项目(ZDYF2020035)。
