PRRSV-N蛋白单抗制备与直接免疫荧光方法建立

Preparation of PRRSV-N Protein Monoclonal Antibody and Establishment of DIF Techniques

为了建立一种快速的PRRSV诊断方法,本研究以纯化的PRRSV-TJ-PGEX-6P-N重组蛋白处理抗原免疫BALB/C小鼠,利用淋巴瘤细胞融合技术,包被带有His标签的PRRSV-N蛋白,通过间接ELISA方法筛选到两株可以稳定分泌的N蛋白特异性单克隆抗体,命名为N-13、N-29。通过间接ELISA方法,WB与IFA方法对其进行鉴定,并测定其亲和常数。腹水效价分别为1:4 000与1:5 000,选择腹水效价高的N-13进行纯化标记,并对标记抗体的特异性、敏感性与保存期进行鉴定;通过DFA条件的摸索,建立了一种敏感的DFA诊断方法,并对其和PRRSV分离鉴定常用的CPE观察法进行比较。结果表明该DFA方法结果准确,可以作为实验室病毒分离鉴定和猪生产中该疾病诊断的有效手段。

In order to establish a rapid diagnostic method for PRRSV, antigen immune BALB/C mice was treated with purified PRRSV-TJ-PGEX-6 P-N recombinant protein. We used lymphoma cell fusion technique, coating the PRRSV-N prote in with His label, to screen 2 specific monoclonal antibodies of N protein by indirect ELISA,named after N-13, N-29. We identified them with indirect ELISA, WB and IFA methods, measuring their affinity constants. Ascites titers were respectively 1： 4 000 and 1： 5 000. We chose N-13 of high ascites titer to tag purification, and identified the specificity, sensibility and storage life of labelled antibody. This study established a sensitive diagnostic method of DFA by exploring conditions of DFA, comparing it with CPE, the common method used in PRRSV isolation and identification. The results showed that DFA assay was accurate, which could be used as an effective approach in the isolation and identification of virus in lab as well as the diagnose of certain disease in pig production.