摘要
目的克隆、表达田鼠巴贝虫(Babesia microti)候选诊断抗原,评价其潜在的诊断价值。方法目的基因来自本室前期构建的田鼠巴贝虫cDNA文库,分别是Bm2、Bm4、Bm6、Bm9和Bm15,用生物信息学分析软件对编码其ORF的氨基酸序列进行预测和分析。从阳性克隆的p Bluscript重组质粒中PCR扩增目的基因。将扩增产物连接至p ET28a质粒,构建重组质粒并转化至大肠埃希菌(Escherichia coli)BL21(DE3)感受态细胞中,用终浓度为1 mmol/L的异丙基-β-D-硫代半乳糖苷(IPTG)进行诱导表达,通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行蛋白表达和可溶性分析。对包涵体表达的蛋白,采用PAGE胶蛋白微量回收试剂盒对目的蛋白进行纯化。用田鼠巴贝虫感染小鼠血清和疟疾患者血清对纯化的重组蛋白进行ELISA分析,评价各重组抗原的敏感性和特异性。结果 5个候选抗原Bm2、Bm4、Bm6、Bm9和Bm15基因经PCR扩增,大小分别约为480、300、750、200和700 bp,与理论值相符。构建的重组质粒经测序鉴定与cDNA文库筛选的阳性克隆的ORF序列一致。Bm4、Bm6和Bm15质粒经IPTG诱导,获得包涵体表达,相对分子质量(Mr)分别为13 000、30 000和30 000。但Bm2和Bm9未能表达成功。3个重组蛋白Bm4、Bm6和Bm15纯化后,经SDS-PAGE电泳分析,获得了单一条带的目的重组蛋白。使用田鼠巴贝虫感染小鼠血清和健康小鼠血清对Bm4、Bm6、Bm15和Bm SA1(参照)重组蛋白的ELISA分析结果显示,重组蛋白Bm4、Bm6、Bm15及Bm SA1的敏感性分别为15.0%、55.0%、80.0%、100.0%,特异性分别为100.0%、100.0%、90.0%、100.0%。Bm4、Bm6及Bm15与疟疾患者血清无交叉反应,Bm SA1与疟疾患者血清有一定的交叉反应(假阳性率为13.3%)。结论表达了3个田鼠巴贝虫候选诊断抗原,初步评价发现Bm15抗原对检测田鼠巴贝虫具有良好的敏感性和特异性。
Objective To clone and express Babesia microti diagnostic candidate antigens, and to preliminarily evaluate their diagnostic value. Methods Immune screening was performed using bioinformatics method on the cDNA library of Babesia microti constructed previously in our lab, resulting in 5 positive clones, namely Bm2, Bm4, Bm6, Bm9 and Bml5. The open reading frames （ORFs） of their amino acid sequences were predicted. The desired genes were amplified using the pBluscript plasmids of positive clones. The products were cloned into pET28a and transformed into E. coli BL21（DE3） system. Expression was induced with isopropyl-13-D-thiogalactopyranoside（IPTG） at a final concentration of 1 mmol/L. Expression and solubility of the recombinant proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）. The insoluble recombinant proteins were purified using Micro Protein PAGE Recovery Kit. The sensitivity and specificity of recombinant antigens were evaluated with ELISA method using mouse sera infected with B. microti and their cross-reactivity with sera of malaria patients wasevaluated. Results The five candidate antigen genes were amplified by PCR and resulted in protein bands of 480, 300, 750, 200 and 700 bp, respectively, as predicted by bioinformaties software. The sequences were verified to be consistent with cDNA library of B. microti. The Bin4, Bin6 and Bml5 genes were induced by IPTG to express as inclusion bodies with relative molecular mass of 13 000, 30 000 and 30 000, respectively. However, expression of Bin2 and Bin9 was not achieved. Bin4, Bin6 and Bml5 proteins were extracted with Micro Protein PAGE Recovery Kit and analyzed by SDS-PAGE, resulting in single protein bands. The sensitivity of Bin4, Bin6, Bml5 and BmSA1 （control） to mouse sera was 15.0%, 55.0%, 80.0%, and 100.0%, respectively, while the specificity was 100.0%, 100.0%, 90.0%, and 100.0%, respectively. Bm4, Bm6 and Bml5 showed no cross-reactivity with sera of malaria patient, whereas BmSA1 showed some positive cross-reactivity with a 13.3% false positive rate. Conclusion Three diagnostic candidate antigens of B. microti are expressed and purified, and Bml5 shows promising sensitivity and specificity as B. microti diagnostics.
作者
刘秀凤
孙嘉慧
徐斌
陈军虎
胡薇
LIU Xiu-feng;SUN Jia-hui;XU Bin;CHEN Jun-hu;HU Wei(Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200438, China;Xi＇an Honghui Hospital, Xi＇an 710054, China;National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, WHO Collaborating Centre for Tropical Diseases, National Center for International Research on Tropical Diseases, Ministry of Science and Technology, Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai 200025, China)
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2017年第6期549-553,共5页
Chinese Journal of Parasitology and Parasitic Diseases
基金
上海市公共卫生体系建设三年行动计划(2015-2017)(No.GWⅣ-29)
卫生行业科研专项经费资助项目(No.201202019)
关键词
田鼠巴贝虫
诊断候选抗原
克隆和表达
评价
Babesia microti
Diagnostic candidate antigens
Cloning and expression
Evaluation
