利用杆状病毒表达系统表达奶牛触珠蛋白及其单克隆抗体的制备

Expression of cow haptoglobin protein using baculovirus expression system and preparation of its monoclonal antibodies

为了建立奶牛早期妊娠诊断检测方法,试验用GenBank公布的奶牛触珠蛋白(haptoglobin)Hp基因序列设计引物,扩增出Hp基因全长片段,将Hp基因亚克隆至Bac-to-Bac昆虫/杆状病毒表达系统的供体质粒pFastBacHTb的多克隆位点中,转入含昆虫杆状病毒骨架质粒的E.coliDH10Bac感受态细胞中,通过蓝白斑筛选获得穿梭质粒rBacmid-Hp阳性克隆,在脂质体介导下转染昆虫细胞sf9,获得含Hp基因的重组杆状病毒质粒rBacmid-Hp;用纯化的rBacmid-Hp蛋白免疫Balb/c小鼠,将免疫小鼠的脾细胞和鼠SP2/0骨髓瘤细胞融合,采用间接ELISA法和有限稀释法筛选杂交瘤细胞;分别用ELISA法、Western-blot检测单克隆抗体腹水效价及特异性。结果表明:在sf9细胞中成功表达Hp蛋白,该蛋白具有良好的生物学活性;制备了8株单克隆抗体,分别命名为3B12、4F4、4F11、5D7、5G8、7E5、10C3和12F9,腹水效价均在1∶5.12×10^4以上;亚类鉴定显示,除3B12、4F11、10C3为IgG2a外,其余单克隆抗体均属于IgG1;筛选出1对配对单克隆抗体,分别为12F9和4F11。说明Hp蛋白可在Bac-to-Bac昆虫/杆状病毒表达系统中高效表达,并成功筛选出一对配对的高效单克隆抗体。

In order to establish a diagnostic method for early pregnancy in dairy cows,haptoglobin ( Hp) gene sequence published in GenBank was adopted in this experiment for primer design. The full length fragment of Hp gene was amplified and was cloned into the polyclonal sites of Bac-to-Bac insect/ baculovirus expression system donor plasmid pFastBacHTb,which was then transformed into DH10Bac competent cells containing baculovirus helper plasmid. After blue-white screening,the shuttle plasmid rBacmid-Hp positive clone was obtained,and was transformed into sf9 cells by liposome. Recombinant baculovirus rBacmid-Hp containing Hp gene was obtained,and the purified rBacmid-Hp protein was immunized to Balb/c mice;spleen cells from immunized mice were fused with mouse SP2 /0 myeloma cells,and indirect ELISA and limited dilution methods were adopted for the screening of hybridoma cells. ELISA and Western-blot methods were used for the determination of ascitic titer and the specificity of mAbs respectively. The results showed that Hp protein was successfully expressed in sf9 cells,with good biological activity. Eight mAbs were prepared and named 3B12,4F4,4F11,5D7,5G8,7E5,10C3 and 12F9 respectively,which had ascitic titers of 1 ∶5.12×10^4 above. Subtype identification showed that except 3B12,4F11,10C3 of IgG2a,the remaining mAbs were all IgG1 . A pair of paired mAbs were screened and named 12F9 and 4F11 respectively. This indicated that Hp protein could be highly expressed in the Bac-to-Bac insect / baculovirus expression system. And one pair of highly effective monoclonal antibodies were successfully screened.