摘要
本研究旨在用杆状病毒/昆虫细胞表达系统表达日本血吸虫热休克70蛋白(SjHSP70),获得重组的rSjHSP70蛋白。利用PCR技术从日本血吸虫cDNA文库中克隆SjHSP70全长序列;亚克隆到pFastBacHTA中,构建重组杆状病毒转移载体pFastBacHTA-SjHSP70,导入DH10Bac感受态细胞,得到重组穿梭质粒reBacmid-SjHSP70,再转染到Sf9昆虫细胞制备重组病毒;将重组病毒感染细胞,用间接免疫荧光技术鉴定重组蛋白的表达;His柱亲和层析法纯化目的蛋白;Western blot技术对纯化蛋白进行抗原性鉴定。SjHSP70基因全长序列1962 bp,在Sf9昆虫细胞中获得高效表达;His柱亲和层析法纯化表达的蛋白可被日本血吸虫感染小鼠的阳性血清识别。用杆状病毒昆虫细胞表达系统成功表达了SjHSP70,为深入研究SjHSP70分子的免疫生物学功能提供了材料。
The objective of the present study was to express recombinant SjHSP70 in insect cells Sf9.The SjHSP70 gene of 1962 bp was amplified by PCR and used to construct vector pFastBacHTA-SjHSP70.Then,reBacmid-SjHSP70 was transformed into competent cells.The recombinant shuttle plasmid reBacmid-SjHSP70 was obtained from the transformed DH10Bac cells and then transfected into insect cells Sf9.The recombinant protein SjHSP70 expressed in infected cells was visualized in IFA and purified with His column affinity chromatography.Western blot demonstrated the ecombinant SjHSP70 was recognized by antiserum from mice infected with Schistosoma japonicum.The availability of recombinant SjHSP70 provided materials for further exploring its immunobiological function.
出处
《中国动物传染病学报》
CAS
2013年第3期45-50,共6页
Chinese Journal of Animal Infectious Diseases
基金
中央级公益性科研院所基本科研业务费项目(2013JB15)
