【摘要】：目的對細粒棘球絳蟲轉(zhuǎn)醛醇酶(Echinococcus granulosus transaldolase,EgTAL)編碼基因進行生物信息學(xué)分析、克隆和表達,并對其作為藥物靶標及其潛在的免疫診斷價值進行初步評價。方法運用多種軟件分析EgTAL的理化性質(zhì)、保守功能域、同源性和三級結(jié)構(gòu)等。從重組質(zhì)粒pBluescript ⅡSK Egtal中PCR擴增Egtal基因,克隆至pET30a,構(gòu)建表達載體pET30a-Egtal,轉(zhuǎn)化大腸埃希菌(Escherichia coli)BL21(DE3),異丙基-β-D-硫代半乳糖苷(IPTG)誘導(dǎo)表達,十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE)分析表達產(chǎn)物,抗His標簽鎳親和層析柱純化重組蛋白EgTAL,與細粒棘球蚴病患者血清進行蛋白質(zhì)印跡(Western blotting)分析。用20份已確診的細粒棘球蚴病患者血清和20份健康者血清,ELISA法評價重組蛋白EgTAL的免疫診斷效果。分光光度法檢測重組蛋白EgTAL的酶活性。結(jié)果 Egtal基因長981 bp,編碼的蛋白含326個氨基酸,理論相對分子質(zhì)量(M_r)為36 332,等電點為5.11,含TAL標識序列DATTNPSLI(31~39 aa)及酶活性催化部位,EgTAL與人TAL的同源性為62%。三級結(jié)構(gòu)分子建模顯示,EgTAL具有A和B兩條完整的蛋白鏈。成功構(gòu)建重組質(zhì)粒pET30a-Egtal。SDS-PAGE和Western blotting分析結(jié)果顯示,重組蛋白EgTAL在E.coli BL21(DE3)中獲得高效表達,在M_r36 332處可見重組蛋白EgTAL條帶,主要以可溶性形式存在,EgTAL可被細粒棘球蚴病患者血清識別。ELISA分析結(jié)果顯示,20份細粒棘球蚴病患者血清和20份健康者血清的平均A_(450)值分別為1.189±0.384和0.325±0.078,其中17份細粒棘球蚴病患者血清檢測結(jié)果為陽性。酶活性檢測結(jié)果顯示,純化后的EgTAL具有高效酶活性,60μg EgTAL加入酶促反應(yīng)體系催化反應(yīng)30 min后,體系的吸光度(A_(340)值)由1.684±0.103降至0.139±0.009。結(jié)論克隆了細粒棘球絳蟲Egtal基因,并在E.coli BL21(DE3)中表達出有酶催化活性和潛在免疫診斷價值的EgTAL重組蛋白。
[Abstract]:Objective to study the bioinformatics analysis, cloning and expression of Echinococcus granulosus transaldolase (Echinococcus transaldolase) encoding gene, and to evaluate the potential value of Echinococcus transaldolase as a drug target and its potential immunological diagnostic value. Methods the physicochemical properties, conserved functional domain, homology and tertiary structure of EgTAL were analyzed by various software. The Egtal gene was amplified from recombinant plasmid pBluescript 鈪，

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