牛传染性鼻气管炎病毒gD蛋白单克隆抗体的制备及鉴定

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

目的制备牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)gD蛋白单克隆抗体,并进行鉴定。方法将扩增的IBRV gD基因插入原核表达载体p ET-28a(+),构建重组质粒gD-pET-28a,转化至感受态E.coli BL21(DE3),经IPTG诱导表达重组蛋白gD,通过Ni-NTA Agarous Kit纯化后,免疫BALB/c小鼠。取小鼠脾细胞,与SP2/0细胞融合,筛选阳性杂交瘤细胞,经小鼠腹腔注射,待小鼠腹腔膨胀时抽取腹水,用Hi Trap Protein G HP试剂盒纯化单抗,进行Western blot及间接免疫荧光检测。结果重组蛋白gD相对分子质量为48 000,纯化后浓度为4.19 mg/ml。小鼠脾细胞与SP2/0细胞融合后筛选出1株阳性杂交瘤细胞株,获得相应单克隆抗体的蛋白含量为1.81 mg/ml,且可与IBRV发生特异性反应,可与接种IBRV的MDBK细胞发生反应,产生特异性荧光。结论本研究利用原核表达系统表达纯化了IBRV gD蛋白,成功制备了IBRV gD单克隆抗体,为下一步表位鉴定及致病机理的研究奠定了基础。

Objective To prepare and identify the monoclonal antibody（Mc Ab） against gD protein of bovine infectious rhinotracheitis virus（IBRV）. Methods The amplified IBRV gD gene was inserted into prokaryotic expression vector pET-28a（＋）. The constructed recombinant plasmid gD-pET-28 a was transformed to E. coli BL21（DE3）and induced with IPTG. The expressed recombinant gD protein was purified by Ni-NTA Agarous Kit, with which BALB/c mice were immunized. The splenocytes of immunized mice were fused with SP2/0 cells, from which positive hybridoma cells were screened and injected i. p. into mice. The ascites of mice were collected, purified by using Hi Trap Protein G HP kit,subclassed and identified by Western blot and indirect immunofluorescence assay. Results The recombinant gD protein with a relative molecular mass of 48 000 reached a concentration of 4. 19 mg/ml after purification. One hybridoma cell strain was screened, and the obtained McAb reached a protein content of 1. 81 mg/ml, and showed specific reaction with IBRV. Specific fluorescence was observed in MDBK cells inoculated with IBRV. Conclusion IBDV gD protein was expressed by prokaryotic expression system and purified, and the Mc Ab against IBDV gD protein was successfully prepared, which laid a foundation of identification of epitope and study on pathogenic mechanism of the virus.