亚洲牛带绦虫肌动相关蛋白基因克隆及表达

Cloning and prokaryotic expression of actin related protein 2/3 complex subunit 4 of Taenia saginata asiatica

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 conduct a preliminary immunoreacticity study on genes of Taenia saginata asiatica ＇s actin related protein 2/3 complex subunit 4 （ Arp2/3 ）. Methods Based on the Arp2/3 sequence template in the library of eDNA of Taenia asiatica, a pair of special primer was synthesized to amplify the gene through PCR technology. The genes were cloned into prokaryotic expression vector Pet-28α（ ＋ ） inducing the target genes to conduct expression through IPTG in competent Escherichia coli BL21/DE3 processed by CaCI2. The products of the expression was identified with SDS-PAGE. As the expression took place in an inclusion body, SKI2 was used to achieve purified recombinant protein and the immunoreacticity study was conducted with Western-blotting. Results PCR,double enzyme digestion and DNA sequen- cing indicated that pET-28a （ ＋ ） and Arp2/3 recombinant plasmid was successfully constructed. SDS-PAGE results showed that the gene expression took place in Escherichia coli BL21/DE3, and highly pure protein was achieved after the dissolving, refolding and particle exchange chromatography of inclusion body deposits. The recombinant protein reacted with Taenia asiatica and taeniarhynchus saginatus infected patients＇ serum, which indicated the immunoreacticity of the protein. Conclusion The Arp2/3 subunit 4 gene is successfully cloned. The recombinant protein is obtained through expression and purification, and the genes immunoreacticity is confmned, which provides the foundation for further studies of the gene.