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目的对细粒棘球绦虫转醛醇酶(Echinococcus granulosus transaldolase,EgTAL)编码基因进行生物信息学分析、克隆和表达,并对其作为药物靶标及其潜在的免疫诊断价值进行初步评价。方法运用多种软件分析EgTAL的理化性质、保守功能域、同源性和三级结构等。从重组质粒pBluescript ⅡSK Egtal中PCR扩增Egtal基因,克隆至pET30a,构建表达载体pET30a-Egtal,转化大肠埃希菌(Escherichia coli)BL21(DE3),异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达产物,抗His标签镍亲和层析柱纯化重组蛋白EgTAL,与细粒棘球蚴病患者血清进行蛋白质印迹(Western blotting)分析。用20份已确诊的细粒棘球蚴病患者血清和20份健康者血清,ELISA法评价重组蛋白EgTAL的免疫诊断效果。分光光度法检测重组蛋白EgTAL的酶活性。结果 Egtal基因长981 bp,编码的蛋白含326个氨基酸,理论相对分子质量(M_r)为36 332,等电点为5.11,含TAL标识序列DATTNPSLI(31~39 aa)及酶活性催化部位,EgTAL与人TAL的同源性为62%。三级结构分子建模显示,EgTAL具有A和B两条完整的蛋白链。成功构建重组质粒pET30a-Egtal。SDS-PAGE和Western blotting分析结果显示,重组蛋白EgTAL在E.coli BL21(DE3)中获得高效表达,在M_r36 332处可见重组蛋白EgTAL条带,主要以可溶性形式存在,EgTAL可被细粒棘球蚴病患者血清识别。ELISA分析结果显示,20份细粒棘球蚴病患者血清和20份健康者血清的平均A_(450)值分别为1.189±0.384和0.325±0.078,其中17份细粒棘球蚴病患者血清检测结果为阳性。酶活性检测结果显示,纯化后的EgTAL具有高效酶活性,60μg EgTAL加入酶促反应体系催化反应30 min后,体系的吸光度(A_(340)值)由1.684±0.103降至0.139±0.009。结论克隆了细粒棘球绦虫Egtal基因,并在E.coli BL21(DE3)中表达出有酶催化活性和潜在免疫诊断价值的EgTAL重组蛋白。
Objective To evaluate the bioinformatics analysis, cloning and expression of the gene encoding Echinococcus granulosus transaldolase (EgTAL), and to evaluate the potential value of EgTAL as a drug target and its potential immunodiagnostic value. Methods Various softwares were used to analyze the physicochemical properties, conserved domains, homologies and tertiary structures of EgTAL. The Egtal gene was amplified by PCR from the recombinant plasmid pBluescript II SK Egtal and cloned into pET30a. The expression vector pET30a-Egtal was constructed and transformed into Escherichia coli BL21 (DE3), isopropyl-β-D-thiogalactopyranoside (IPTG) induced expression, the expression product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the recombinant protein EgTAL was purified by anti-His tag nickel affinity chromatography, Patient sera were analyzed by Western blotting. 20 cases of diagnosed patients with Echinococcus granulosus serum and 20 healthy serum, ELISA method to evaluate the immune diagnosis of recombinant protein EgTAL. The enzymatic activity of the recombinant protein EgTAL was detected spectrophotometrically. RESULTS: The Egtal gene was 981 bp in length and encoded a protein of 326 amino acids. The theoretical relative molecular mass (M_r) was 36 332 and the isoelectric point was 5.11. The ETA gene contained the TAL-tagged DATTNPSLI (31-39 aa) and catalytic sites of EgTAL Homology with human TAL was 62%. Molecular modeling of tertiary structure shows that EgTAL has two complete protein chains, A and B. The recombinant plasmid pET30a-Egtal was successfully constructed. The results of SDS-PAGE and Western blotting showed that the recombinant protein EgTAL was highly expressed in E.coli BL21 (DE3), and the recombinant protein EgTAL band was seen at M_r36 332, mainly in soluble form. Echinococcosis patients serum identification. The results of ELISA showed that the average A_ (450) values of sera from 20 Echinococcus granulosus patients and 20 healthy controls were 1.189 ± 0.384 and 0.325 ± 0.078, respectively. Serum samples from 17 patients with Echinococcus granulosus The result is positive. The results of enzymatic activity showed that the purified EgTAL had high enzyme activity. The absorbance (A_ (340) value) of the system was decreased from 1.684 ± 0.103 to 0.139 ± 0.009 after 60 μg EgTAL was added into the enzymatic reaction for 30 min. Conclusion The Egtal gene of Echinococcus granulosus was cloned and expressed in E.coli BL21 (DE3) with the enzymatic activity and potential immunodiagnostic value of EgTAL recombinant protein.