一种犬细小病毒基因工程抗体的制备

doi:10.16431/j.cnki.1671-7236.2019.10.026

摘要/Abstract

摘要： 本研究旨在利用HEK-293细胞系制备鼠源犬细小病毒（canine parovirus，CPV）基因工程抗体并检测其生物活性。通过抗体亚型检测试剂盒检测CPV单克隆抗体亚型；采用间接ELISA检测CPV单克隆抗体的亲和力和特异性；经RACE-PCR获得CPV单克隆抗体的可变区序列，将可变区序列与鼠源抗体恒定区序列连接；分别构建真核表达载体pcDNA3.1（+）-L和pcDNA3.1（+）-H，将载体共转染HEK-293细胞，采用血凝抑制与中和试验的方法检测鼠源CPV基因工程抗体生物活性；采用HEK-293F细胞悬浮表达并用间接ELISA方法检测鼠源CPV基因工程抗体的表达量；用Protein A亲和层析柱纯化鼠源CPV基因工程抗体后进行SDS-PAGE鉴定；间接免疫荧光检测纯化后鼠源CPV基因工程抗体的活性。结果显示，CPV单克隆抗体亚型为IgG_2b，亲和力常数6个Ka平均值为1.02×10¹¹ L/mol，只与CPV VLPs发生反应。琼脂糖凝胶电泳结果显示，试验成功构建真核表达载体pcDNA3.1（+）-L和pcDNA3.1（+）-H；HEK-293和HEK-293F细胞培养上清液血抑效价分别为1∶2⁴和1∶2⁶，中和试验结果显示，HEK-293和HEK-293F细胞培养上清液中和效价分别为1∶152和1∶1 290；鼠源CPV基因工程抗体在HEK-293F细胞中的表达量为5.97 mg/L，SDS-PAGE分析在55和25 ku处出现条带，表明鼠源CPV基因工程抗体成功在HEK-293F细胞中表达并纯化。间接免疫荧光检测结果表明，纯化后鼠源CPV基因工程抗体具有良好的生物活性。本研究在HEK-293F细胞中成功表达具有中和活性、纯度较高的鼠源CPV基因工程抗体，为今后CPV基因工程抗体药物的研发奠定基础。

关键词: 犬细小病毒(CPV); 基因工程抗体; 共转染; HEK-293F细胞

Abstract: The aim of this study was to prepare a mouse genetically engineered antibody against canine parvovirus (CPV) using HEK-293 cell line and to detect its biological activity.The CPV monoclonal antibody subtype was detected by antibody subtype detection kit.The affinity and specificity of CPV monoclonal antibody were detected by indirect ELISA,the variable region of CPV monoclonal antibody was obtained by RACE-PCR,and linking the variable region sequence to the murine antibody constant region sequence.The eukaryotic expression vectors of pcDNA3.1(+)-L and pcDNA3.1(+)-H were constructed,respectively.The expression vectors was co-transfected into HEK-293 cells,and the biological activity of mouse CPV genetically engineered antibody was detected by hemagglutination inhibition techniques and neutralization testing.The mouse CPV genetically engineered antibody was expressed by using HEK-293F cells and the expression of mouse CPV genetically engineered antibody in HEK-293F cells was detected by indirect ELISA.The mouse CPV genetically engineered antibody was purified by Protein A affinity chromatography column and detected by SDS-PAGE.The activity of the purified mouse CPV genetically engineered antibody was detected by indirect immunofluorescence.The results showed that CPV monoclonal antibody was belong to IgG_2b,and the average of affinity constant of 6 Ka was 1.02×10¹¹ L/mol,which only reacted with CPV VLPs.The results of agarose gel electrophoresis showed that pcDNA3.1(+)-L and pcDNA3.1(+)-H were successfully constructed,and the hemagglutination inhibition titer of culture supernatant of HEK-293 and HEK-293F cells were 1:2⁴ and 1:2⁶,respectively;The neutralization experiments showed that the neutralization titer of culture supernatant of HEK-293 and HEK-293F cells were 1:152 and 1:1 290,respectively;The expression of mouse CPV genetically engineered antibody in HEK-293F cells was 5.97 mg/L.SDS-PAGE analysis showed bands at 55 and 25 ku.The results showed that the mouse CPV genetically engineered antibody was successfully expressed and purified in HEK-293F cells.Indirect immunofluorescence assay showed that the purified mouse CPV genetically engineered antibody had good biological activity.In this study,we successfully expressed mouse CPV genetically engineered antibody with high neutralizing activity and high purity in HEK-293F cells,it yet laid a foundation for study of CPV genetically engineered antibody drugs in the future.

Key words: canine parvovirus(CPV); genetically engineered antibody; co-transfected; HEK-293F cell

中图分类号:

S852.65⁺5

李希辰, 雷欢, 李雪, 石晶, 殷玉和, 吴丛梅. 一种犬细小病毒基因工程抗体的制备[J]. 中国畜牧兽医, 2019, 46(10): 3042-3051.

LI Xichen, LEI Huan, LI Xue, SHI Jing, YIN Yuhe, WU Congmei. Preparation of a Canine Parvovirus Genetically Engineered Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(10): 3042-3051.

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