肝片吸虫鞘脂激活蛋白样蛋白2(FhSAP-2)的原核表达及分离纯化

PROKARYOTIC EXPRESSION AND PURIFICATION FOR F.HEPATICA SAPOSIN- LIKE PROTEIN 2( FhSAP- 2)

目的建立肝片吸虫鞘脂激活蛋白样蛋白2(FhSAP-2)原核表达系统。方法用生物信息学软件OptimumTMCodon优化密码子适应指数(CAI)、最优密码子频率(FOP)及GC含量以获得适合大肠杆菌BL-21表达的FhSAP-2基因序列,并克隆到原核表达载体pET22b和pET28a中,经IPTG诱导其表达并经亲和层析及离子交换色谱纯化后获得目的蛋白。结果 OptimumTMCodon获得优化后的FhSAP-2序列,经测序及酶切鉴定结果表明重组质粒pET22b-FhSAP-2、pET28a-FhSAP-2构建成功,进一步实验结果表明FhSAP-2在pET28a中包涵体部位表达,可溶部位不表达;在pET22b中可溶部位表达量低,包涵体部位无表达。继而经Ni-NTA柱亲和纯化及离子交换色谱纯化获得电泳纯的FhSAP-2蛋白。结论成功建立FhSAP-2的原核表达系统。

Objective Constructed a prokaryotic expression system for Fasciola hepatica saposin like protein 2（FhSAP-2）.Method Bioinformatics software OptimumTM Codon was used to obtain a suitable sequence of FhSAP-2 gene expressed in E.coli BL-21 by optimizing of codon adaptation index（CAI）,the optimal codon frequency（FOP）and the content of GC.And the optimization sequence was cloned into prokaryotic expression vector pET-22b and pET-28a.IPTG induction,Ni-NTA affinity chromatography and ion exchange chromatography were utilized to obtain FhSAP-2 protein.Results We procure SAP-2 sequence suitable for prokaryotic expression system by OptimumTM Codon software. Recombinant plasmids pET22b-FhSAP-2 and pET28a-FhSAP-2 were constructed successfully and identified by double-enzyme digested and sequencing.Further,our results were shown that FhSAP-2 was expression in inclusion bodies part of pET28a-FhSAP-2 but not in pET22b-FhSAP-2.Finally we obtained high-purity FhSAP-2 protein by affinity chromatography and ion exchange chromatography.Conclusions This study has been successfully established the prokaryotic expression system of FhSAP-2.