摘要
通过BL21（DE3）原核表达系统制备抗猪流行性腹泻病毒（PEDV）猪源化单链抗体scFv-Fc。提取抗PEDV杂交瘤细胞2D1的总RNA，通过反转录与SOE-PCR方法获得单链抗体scFv基因，同时提取猪脾脏组织的总RNA，RT-PCR获得猪免疫球蛋白恒定区Fc基因。通过SOE-PCR方法将scFv与Fc连接获得猪源性单链抗体scFv-F（融合基因，构建重组质粒pET-28a-scFv-Fc，将该重组质粒转入BL21（DE3）原核表达茵进行诱导表达，SDS-PAGE分析及Westernblot检测融合蛋白的特异性，再对表达蛋白进行纯化和复性，通过间接ELISA鉴定融合蛋白与PEDV的结合活性。结果显示，插入pET-28a载体的scFnFc序列长度1128bp，抗PEDV猪源化单链抗体的相对分子质量为45800，重组蛋白具有较高的特异性。本研究成功构建了pET-28a-scFv-Fc原核表达系统，重组蛋白具有较高的免疫反应性，为PEDV重组抗体的研究奠定理论基础。
To construct porcine anti PEDV single-chain fragment antibody（scFwFc） through BL21 （DE3） prokaryotic expression system. The total RNA of the anti PEDV hybridoma cell line 2D1 was extracted,and single-chain fragment antibody scFv was obtained by reverse transcription and SOE-PCR. The total RNA of the porcine spleen tissue was extracted,and the pig immunoglobulin constant region Fc gene was obtained by reverse transcription and PCR. Then the scFv and Fc were connected by SOE-PCR. The scFv-Fc gene was cloned into the prokaryotic expression vector pET-28a. The recombinant expression vector pET-28a-scFv-Fc was constructed and transformed into E. coli BL21 （DE3） ,and recombinant protein was obtained. The purification and renaturation were done,and SDS-PAGE analysis was carried out. Then the expression of the fusion protein was detected by Western blot assay and the binding activity of fusion proteins with PEDV was identified by indirect LISA. The length of fusion gene scFv-Fc sequence was 1 128 bp. The molecular weight of the recombinant expression proteins of porcine anti PEDV single antibody was 45 800. The recombinant proteins showed high specificity with HRP anti-labeled 6 X His IgG antibody. This experiment successfully constructs pET-28a-scFv-Fc prokaryotic expression system of re- combinant protein with high immune reactivity, which provides the basis of further study of anti PEDV recombinant antibodies.
作者
刘慧敏
常琛
尹志安
朱文姣
周国丽
杨国庆
LIU Hui-min;CHANG Chen;YIN Zhi-an;ZHU Wen-jiao;ZHOU Guo-li;YANG Guo-qing(College of Life Sciences,Henan Agricultural University,Zhengzhou 450002,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2018年第8期1467-1472,共6页
Chinese Journal of Veterinary Science
基金
河南省高等学校重点科研资助项目(16A180005)
河南农业大学青年创新基金资助项目(30601444)
