摘要
将IBV H120毒株通过接种鸡胚的方法扩繁,从含病毒的尿囊液中提取总RNA,RT-PCR扩增N基因,构建原核表达载体pGEX-6p-1-N.将测序正确的重组质粒转化入E.coli Transetta(DE3)感受态,对重组蛋白进行诱导表达并纯化,切除GST标签.采用纯化的N蛋白制备抗原免疫BALB/c小鼠,取免疫效果最好的小鼠B淋巴细胞与SP2/0细胞进行融合.利用间接ELISA方法筛选阳性杂交瘤细胞,经过3次亚克隆筛选出13株单抗,其中8株能与IBV结合.通过体内诱生腹水的方法对1A12C5细胞株进行大量制备,辛酸/硫酸铵方法对腹水进行纯化并鉴定.
IBV H120 strain was expanded by inoculation of chicken embryos. And total RNA was extracted from virus-containing allantoic fluid. N gene was amplificated by RT-PCR. Prokaryotic expression vector pGEX-6 p-1-N was constructed. The correct recombinant plasmid was transformed into the E.coli Transetta（DE3） competent cells. The recombinant plasmid was induced and expressed. The recombinant N protein was purified and GST tag was excised. Purified N protein was used to immunize BALB/c mice. B lymphocyte of mice with the best immune effect was selected for cell fusion. Positive hybridoma cells were selected by indirect ELISA. Thirteen hybridoma cell lines that stably produced anti-N protein antibodies were obtained after three rounds of subcloning. Eight hybridoma cell lines of them were able to combine with IBV. The mAb was prepared in large quantities by generate ascites fluid. The ascites was purified by the caprylic/ammonium sulfate and identified.
作者
周景明
耿玥
马文利
刘红亮
祁艳华
张改平
王爱萍
ZHOU Jingming;GENG Yue;MA Wenli;LIU Hongliang;QI Yanhua;ZHANG Gaiping;WANG Aiping(School of Life Sciences,Zhengzhou University,Zhengzhou 450001,China)
出处
《郑州大学学报（理学版）》
CAS
北大核心
2018年第4期75-79,共5页
Journal of Zhengzhou University:Natural Science Edition
基金
国家重点研发计划项目(2016YFD0500800)
