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目的:制备人B组轮状病毒WH-2株vp7基因的单克隆抗体(mAb),并研究其免疫学特性。方法:将B组轮状病毒WH-2株vp7基因克隆至pGEX-KG载体,然后转入大肠杆菌E.coliTop10,IPTG诱导表达GST-VP7融合蛋白。分离纯化蛋白GST-VP7并免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞Sp2/0融合,利用间接ELISA法筛选阳性杂交瘤细胞,有限稀释法克隆化,获得稳定分泌mAb检测为阳性的细胞株。结果:经过3次克隆化筛选,最终得到1株能和GST-VP7蛋白反应稳定分泌mAb的阳性细胞克隆,这些mAb通过Western blot鉴定结果显示其具有良好的特异性。结论:人B组轮状病毒WH-2株VP7蛋白成功在大肠杆菌中GST融合表达及mAb的制备体能用于GBRV结构和功能的研究,也为其引起的疾病预防、诊断和治疗等研究和临床诊断奠定了基础。
OBJECTIVE: To prepare monoclonal antibodies (mAbs) for the vp7 gene of human rotavirus B group WH-2 and study its immunological properties. Methods: The vp7 gene of group B rotavirus WH-2 was cloned into pGEX-KG vector and then transformed into E. coli E.coli TOP10. IPTG was used to induce the expression of GST-VP7 fusion protein. The purified protein GST-VP7 was isolated and immunized BALB / c mice were spleen cells and Sp2 / 0 fusion of myeloma cells, using indirect ELISA screening of positive hybridoma cells, limiting dilution cloning, stable secretion of mAb was positive Of cell lines. Results: After 3 times of cloning and screening, we got 1 positive cell clones that can react with GST-VP7 protein to secrete mAb stably. These mAbs were proved to have good specificity by Western blot. CONCLUSIONS: The fusion protein of human B-2 with VP-B of WH-2 strain in E. coli and the preparation of mAb can be used to study the structure and function of GBRV and its prevention, diagnosis and treatment Clinical diagnosis laid the foundation.