牛传染性鼻气管炎病毒gE蛋白单克隆抗体的制备及鉴定

Preparation and identification of monoclonal antibody against gE protein of bovine infectious rhinotracheitis virus

目的制备牛传染性鼻气管炎病毒(infectious bovine rhinotracheities virus,IBRV)g E蛋白的单克隆抗体,并进行鉴定。方法将扩增的IBRV gE基因插入至载体p ET-32a(+),转化感受态E.coli BL21(DE3),经IPTG诱导表达重组蛋白IBRV gE,通过Ni-NTA Agarous Kit纯化后,免疫BALB/c小鼠。取免疫小鼠脾细胞,与SP2/0细胞融合,间接免疫荧光法筛选阳性杂交瘤细胞。经小鼠腹腔注射杂交瘤细胞,3×10~6个/只,待小鼠腹腔膨大后,采集腹水,硫酸铵沉淀法纯化单克隆抗体,并进行亚类、浓度、染色体数目、Western blot及间接免疫荧光检测。结果重组蛋白IBRV gE相对分子质量约35 000,纯化后浓度为1.2 mg/mL。小鼠脾细胞与SP2/0细胞融合后,筛选出1株稳定分泌IBRV单抗的杂交瘤细胞(命名为3G9),获得相应抗体浓度为1.46 mg/m L;为IgG1亚类,轻链为kappa链;染色体数目为95~105;可与IBRV发生特异性反应;可与接种IBRV毒株的MDBK细胞发生特异性结合,产生特异性荧光。结论采用原核表达系统表达并纯化了IBRV gE蛋白,成功制备了抗IBRV gE的单克隆抗体,为建立特异性的IBRV诊断方法及致病机理的研究奠定了基础。

Objective To prepare and identify the monoclonal antibody （McAb） against gE protein of bovine infectious rhinotracheitis virus （IBRV）. Methods IBRV gE gene was amplified and inserted into vector pET-32a （＋）, and the constructed recombinant plasmid was transformed to E. coli BL21 （DE3） and induced with IPTG. The expressed recombinant protein IBRV gE was purified with Ni-NTA Agarous Kit, with which BALB/c mice were immunized. The splenocytes of immunized mice were fused with SP2/0 cells, and the positive hybridoma cells were screened by indirect immu- nofluorescence assay （IFA）. Mice were injected i. p. with the hybridoma cells, 3 x 106 cells for each, of which the ascites were collected. MeAb was purified from the ascites by precipitation with ammonium sulphate and subclassed, the determined for concentration and chromosome number, and identified by Western blot and IFA. Results The recom- binant protein IBRV gE, with a relative molecular mass of about 35 000, reached a purity of 1.2 mg/mL after puri- fication. A hybridoma cell strain stably secreting McAb was screened and named as 3G9, and the concentration of obtained McAb was 1. 46 mg/mL. The McAb belonged to IgG1 subgroup with kappa light chain and 95 - 105 chro- mosomes, which showed specific reaction with IBRV. The McAb showed specific binding to the MDBK ceils inoculated with IBRV, producing specific fluorescence. Conclusion IBDV gE protein was expressed by prokaryotic expression system and purified, and the McAb was prepared, which laid a foundation of development of specific diagnostic method and study on pathogenic mechanism of IBRV.