摘要
目的重组并原核表达人乙酰肝素酶(HPA),并对重组产物r HPA进行免疫原性鉴定,获得足够的乙酰肝素酶为其功能研究奠定基础。方法利用包含HPA全长序列质粒为模板,扩增肝素酶c DNA片段,将其插入表达载体p ET42a,转化到大肠杆菌BL21DE3中,0.5 mmol/L的IPTG诱导表达。SDS-PAGE检测目的重组蛋白表达情况,NiNTA亲和层析法提纯r HPA,Western Blot和ELISA检测r HPA的免疫原性。结果成功构建了人乙酰肝素酶原核表达系统,r HPA表达量为细菌总蛋白的23.4%,SDS-PAGE显示提纯后的r HPA为单一的蛋白条带。鼠抗人HPA能有效识别r HPA。结论所构建的人乙酰肝素酶原核表达系统能高效表达r HPA,表达的产物有良好的免疫原性。
Objective Recombinant and prokaryotic expression were conducted on human heparanase（ HPA）,and the immunogenicity identification on the r HPA of recombinant product,so as to get enough heparanase to lay the foundation for the study of its function. Methods Using the full length HPA plasmid as template,the c DNA fragment of HPA was amplified,and the expression of p ET42 a was added into p ET42 a vector and transformed into E. coli BL21DE3,and then induced for the expression by 0. 5 mmol / L of IPTG. SDS- PAGE was used to measure the expression of target recombinant protein,Ni- NTA affinity chromatography was applied to extract r HPA. Western blot and ELISA were applied to identify immunogenicity of r HPA. Results The HPA prokaryotic expression systems was successfully built,the expression of r HPA was approximately 23. 4% of the total bacterial proteins. The extracted r HPA showed a single protein band in gel after SDS- PAGE. The expression output of r HPA reacted with anti- heparanase antibody. Conclusion The constructed HPA prokaryotic expression system enables to express r HPA with high efficiency,and r HPA has good immunological characteristics of heparanase.
出处
《中国卫生检验杂志》
CAS
2015年第24期4292-4294,共3页
Chinese Journal of Health Laboratory Technology
基金
浙江省医药卫生科学研究基金(2013KYA047)
关键词
乙酰肝素酶
重组表达
免疫原性
Heparanase
Recombinant expression
Immunogenicity
