Expression and evaluation of diagnostic candidate antigens from Babesia microti

Abstract

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

CLC Number:

R382.9

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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References 31

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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