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目的对亚洲牛带绦虫(Taenia saginata asiatica)成虫肌动相关蛋白2/3复合体亚单位4(Actin relatedprotein2/3complex subunit4)基因进行克隆、表达和免疫学研究。方法以亚洲牛带绦虫cDNA文库中肌动相关蛋白2/3复合体亚单位4的已知序列设计合成一对特殊引物,进行PCR技术扩增目的基因,克隆到原核表达载体pET-28a(+)中,在CaCl2制备的感受态大肠埃希菌BL21/DE3中经过异丙硫代-β-D半乳糖苷(IPTG)诱导目的基因表达,表达产物经过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行鉴定。由于蛋白在包涵体表达,故通过N-十二烷基肌氨酸钠(SKL)法纯化获得纯化重组蛋白并用蛋白印迹(Western-blotting)进行免疫学分析。结果PCR、双酶切及DNA测序结果均表明,pET-28a(+)-Arp2/3重组质粒构建成功。SDS-PAGE结果表明,基因在大肠埃希菌BL21/DE3包涵体中表达,经过包涵体沉淀的溶解、重折叠和离子交换层析方法获得高纯度蛋白。重组蛋白能识别亚洲牛带绦虫患者及牛带绦虫患者血清,表明该蛋白具有免疫反应性。结论成功克隆亚洲牛带绦虫肌动相关蛋白2/3复合体亚单位4基因,表达和纯化得到了该基因的重组蛋白并且证明该基因具有免疫反应性,为进一步研究该基因的功能提供条件。
Objective To clone, express and immunologically study the Actin related protein 2/3 complex subunit 4 gene from adult Taenia saginata asiatica. Methods A pair of special primers was designed and synthesized based on the known sequence of actin-related protein 2/3 complex subunit 4 in the Taenia saginata cDNA library. The target gene was amplified by PCR and cloned into prokaryotic expression vector pET-28a (+ ), The expression of the target gene was induced by isopropylthio-β-D-galactoside (IPTG) in the competent Escherichia coli BL21 / DE3 prepared by CaCl2. The expression product was purified by sodium dodecyl sulfate-polypropylene Amide gel electrophoresis (SDS-PAGE) were identified. Since the protein was expressed in inclusion bodies, the purified recombinant protein was purified by N-sodium lauryl sarcosine (SKL) method and subjected to immunological analysis by Western-blotting. Results PCR, double enzyme digestion and DNA sequencing showed that pET-28a (+) - Arp2 / 3 recombinant plasmid was successfully constructed. The results of SDS-PAGE showed that the gene was expressed in E. coli BL21 / DE3 inclusion bodies, and the high purity protein was obtained through the dissolution, refolding and ion exchange chromatography of inclusion body precipitation. The recombinant protein can recognize the serum of patients with Taenia saginata and Taenia saginata asiatica, indicating that the protein is immunoreactive. Conclusion The 4 gene of actinoid 2/3 complex of Taenia saginata Asia has been cloned successfully. The recombinant protein of this gene has been obtained by expression and purification, and the gene has been proved to be immunoreactive, which may provide conditions for further study on the function of this gene.