关键词: 细粒棘球绦虫, 转醛醇酶基因, 克隆表达, 免疫诊断, 药物靶点

Abstract:

Objective To perform bioinformatics analysis of the gene encoding Echinococcus granulosus transaldolase(EgTAL), clone and express this gene, and investigate the role of EgTAL as a drug target for anti-hydatid treatment and the diagnostic value. Methods Multiple softwares were employed to analyze the physical and chemical properties, the conserved domain, phylogenetics, and tertiary structure of EgTAL. Egtal gene was amplified and cloned into vector pET30a. The constructed recombinant plasmid pET30a-Egtal was transformed into E.coli BL21 (DE3) for expression under the induction of IPTG. The expressed product was purified with His60 Ni Superflow affinity chromatography and identified by SDS-PAGE and Western blotting in the presence of serum of patient with cystic echinococcosis. The diagnostic value of EgTAL recombinant protein was assessed by ELISA in sera from 20 patients with cystic echinococcosis and from 20 healthy individuals. The enzyme activity of EgTAL was determined by spectrophotometry. Results The Egtal gene showed a length of 981 bp, encoding a 326-amino-acid protein with a predicted Mr of 36 332 and an isoelectric point (PI) of 5.11. The EgTAL protein contained a TAL-specific sequence DATTNPSLI (31-39 aa) and a key catalytic site. The analysis of phylogenetic tree revealed that the homology between EgTAL and human TAL was 62%. The tertiary structure of EgTAL had two complete protein chains, A and B. The pET30a-Egtal recombinant plasmid was constructed. The results of SDS-PAGE and Western blotting indicated that the recombinant protein EgTAL was highly expressed as a soluble protein in E. coli BL21(DE3) with an Mr of 36 332, and can be recognized by serum from patient with cystic echinococcosis. ELISA indicated that the average A450 value of sera from the cystic echinococcosis patients and healthy people were 1.189±0.0384 and 0.325±0.078, respectively, and 17 out of 20 serum samples from the patients showed positive results.The enzyme activity assay revealed that the purified EgTAL recombinant protein had a significant enzyme activity. At 30 min after addition of 60 μg EgTAL into the enzymatic reaction system, the A340 value dropped to 0.139±0.009 from 1.684±0.103.  Conclusion Egtal gene is cloned and recombinant protein EgTAL is highly expressed in E. coli BL21(DE3). The protein EgTAL shows an enzyme activity and potential immunodiagnostic value.

Key words:  Echinnococcus granulosus, Transaldolase gene, Cloning and expression, Immunodiagnosis, Drug target