禽呼肠孤病毒σ3蛋白单克隆抗体的制备及特性鉴定

Preparation and Identification of Monoclonal Antibody of Protein σ3 from Avian Reovirus

用体外表达的蛋白作为免疫原来制备特异的单克隆抗体,并对制备的抗体进行鉴定。将ARVσ3蛋白基因在体外扩增,扩增产物与载体PGEX-4T-1连接并在基因工程菌中表达,体外表达的蛋白经纯化后作为免疫原来制备特异的单克隆抗体和ELISA检测包被抗原,测定纯化后蛋白的浓度,按每鼠100μg的蛋白用量免疫BALB/c小鼠,免疫4次后取其脾细胞与骨髓瘤细胞SP2/0按5∶1进行融合。对融合后的杂交瘤细胞及时筛选,阳性孔经3次有限稀释法克隆,通过间接E-LISA方法测定其抗体效价,并通过Western Blot、Dot-ELISA、直接免疫荧光、病毒中和等方法对获得的单抗特性进行检测。结果通过纯化的病毒含量为41.5 mg/mL,应用纯化的病毒免疫BALB/c小鼠后与骨髓瘤细胞融合,通过克隆筛选成功获得1株能稳定传代并分泌抗禽呼肠孤病毒单克隆抗体的杂交瘤细胞株σ3 B6-3 K3,用其制备的腹水经间接E-LISA测定效价达105以上,并且与其他参试病毒株没有交叉反应,具有良好的特异性;病毒中和试验证明其中和能力低。应用ARVσ3蛋白制备并获得的杂交瘤细胞株σ3 B6-3 K3具有很好的特异性,不具有中和ARV的关键表位,可以用于ARV的特异性检测。

The specific monoclonal antibody was made using expression protein σ3 from purified competent cell and the Characteristics was detected.The ARV σ3 genome was amplified by RT-PCR.Then the PCR product was ligated with the express vector PGEX-4T-1 and transferred into E.coli.The concentration was test by protein testing Kit after purification the protein σ3.This protein could use for ELISA test and immunization.BALB /c mice were regularly immunized with purified protein σ3 contain 100 μg.After injecting four times,the spleen cells were collected and infused with myeloma cell sp2/0 follow ratio 5 ∶ 1.After selection in time and 3 times clone by limiting dilution.The monoclonal antibody was detected by indirect ELISA,dot-ELISA,immunofluorescence test,and virus neutralization reaction.The results showed that the purification protein was 41.5 mg /mL.One hybridoma cells lines against reovirus named σ3 B6-3 K3 was obtained.The titles were over 105 by indirect ELISA detection.It was not cross with others virus strains in this test by dot-ELISA.It have the lower title by neutralization with reovirus S1733.It indicated that the hybridoma cell lines possessed the favorable specificity.It is not the epitope of avian reovirus.It could be used to detecting avian reovirus in the future.