田鼠巴贝虫候选诊断抗原的表达和评价

摘要/Abstract

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pBluscript重组质粒中PCR扩增目的基因。将扩增产物连接至pET28a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（M_r）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BmSA1（参照）重组蛋白的ELISA分析结果显示,重组蛋白Bm4、Bm6、Bm15及BmSA1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,BmSA1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。

关键词: 田鼠巴贝虫, 诊断候选抗原, 克隆和表达, 评价

Abstract:

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 Bm15.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-β-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 was evaluated.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 bioinformatics software.The sequences were verified to be consistent with cDNA library of B.microti.The Bm4, Bm6 and Bm15 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 Bm2 and Bm9 was not achieved.Bm4, Bm6 and Bm15 proteins were extracted with Micro Protein PAGE Recovery Kit and analyzed by SDS-PAGE, resulting in single protein bands.The sensitivity of Bm4, Bm6, Bm15 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 Bm15 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 Bm15 shows promising sensitivity and specificity as B.microti diagnostics.

Key words: Babesia microti, Diagnostic candidate antigens, Cloning and expression, Evaluation

中图分类号:

R382.9

刘秀凤, 孙嘉慧, 徐斌, 陈军虎, 胡薇. 田鼠巴贝虫候选诊断抗原的表达和评价[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(6): 549-553.

Xiu-feng LIU, Jia-hui SUN, Bin XU, Jun-hu CHEN, Wei HU. Expression and evaluation of diagnostic candidate antigens from Babesia microti[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(6): 549-553.

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引物名称 Primer name	核酸序列5′→3′ Nucleic acid sequence 5′→3′
Bm2F	AATGGATCCATGAAGTCAGTTAGACCAATACTAAT
Bm2R	AATCTCGAGTTTCATACTCAAAATTTTCAGCA
Bm4F	AATGGATCCATGGGATATTTGACTGAGATATACA
Bm4R	AATCTCGAGGAAATCTGCGATGTGAATATGT
Bm6F	AATGGATCCATGGTGTCATTTAAATCAATATTAGT
Bm6R	AATCTCGAGATAACCTTGGATTTGTGTTCTTC
Bm9F	AATGGATCCATGTTTTATTACGGTTTAGGCG
Bm9R	AATCTCGAGTCTATCAGAAAACATGCCATGA
Bm15F	AATGGATCCATGATGAAAATTAACATAGATAAGAT
Bm15R	AATCTCGAGAATACAAACTGGAGTATTTACAGAC

阳性克隆Clone	编码的氨基酸Amino acids	相对分子质量M_r	等电点pI	信号肽Signal peptide	跨膜区Transmembrane domain	结构域 Domain
Bm2	160	18 164	4.81	有 Yes	无 None	低复杂性 Low complexity
Bm4	102	11 495	4.64	无 No	无 None	低复杂性 Low complexity
Bm6	251	28 610	4.85	有 Yes	无 None	卷曲螺旋 Coiled coil
Bm9	60	6 858	3.93	无 No	2(2~24, 39~58)	跨膜区域 Transmembrane region
Bm15	244	28 917	5.47	有 Yes	无 None	低复杂性 Low complexity