绵羊早孕因子单克隆抗体的制备及鉴定

发布时间：2018-04-27 23:49

  本文选题：绵羊 + 早孕因子　； 参考：《河北农业大学》2015年硕士论文

【摘要】：早孕因子(Early pregnancy factor,EPF)是母体受精后从血清中检测出最早与妊娠相关的蛋白,是与妊娠相关的免疫抑制因子,被认为是最早能确认妊娠的生化指标之一。在养殖生产中,利用早孕因子的特异性可以及时进行绵羊的妊娠诊断,减少其空怀发生,为提高生产效率提供重要意义。同时,EPF还可以跟踪妊娠母体中胎儿的发育情况,鉴定胚胎移植效果。目的:应用分子生物和细胞融合技术制备了绵羊早孕因子重组蛋白和单克隆抗体,为进一步研制早孕因子试剂盒奠定基础。方法:对表达菌p GEX4t-EPF-BL21(DE)进行IPTG诱导剂,对诱导剂IPTG进行条件优化。将表达出的绵羊早孕因子重组蛋白(EPF-GST),通过亲和吸附层析柱对蛋白进行纯化,并用SDS-PAGE进行鉴定。以纯化后的EPF重组蛋白为抗原免疫Balb/c小鼠,免疫3次,每次免疫时间间隔为15天,后采用间接ELISA方法检测小鼠效价,取效价合格小鼠(N/P"g2)的脾细胞,将脾细胞和骨髓瘤细胞进行细胞融合,对杂交瘤细胞进行ELISA方法进行筛选,筛选出阳性杂交瘤细胞和细胞株。经有限稀释法将筛选的细胞株注入小鼠腹腔,用G-sepharose柱亲合层析法对小鼠腹水型抗体进行纯化,应用SDS PAGE和Western blot分析抗体纯化后的纯度及特异性。结果:在37℃下,表达菌最佳优化条件为:IPTG浓度0.5mmol/m L,诱导时间4h,在此诱导条件下表达菌能最大量表达绵羊EPF重组蛋白,重组蛋白分子量大小为26KD(其中早孕因子蛋白为10KD,标签蛋白为16KD),且该表达重组蛋白以可溶性形式存在。用纯化后的重组蛋白免疫小鼠,小鼠血清效价可达1:16000。取小鼠的脾细胞与骨髓瘤细胞融合,得到了1株抗绵羊EPF单克隆抗体的细胞株B4D5,效价为1:1000。腹水型抗体经G-sepharose柱亲合层析,SDS PAGE和Western blot检测,得到纯度较高的单克隆抗体,且与绵羊EPF具有一定的特异性。结论:从表达菌中制备出了绵羊EPF重组蛋白(EPF-GST),以重组蛋白免疫小鼠,得到了绵羊早孕因子单克隆抗体。
[Abstract]:Early pregnancy factor EPFs are the earliest pregnancy-related proteins and immunosuppressive factors associated with pregnancy after maternal fertilization, and are considered to be one of the earliest biochemical markers to identify pregnancy. The early pregnancy factor can be used to diagnose the pregnancy of sheep in time, reduce the occurrence of empty pregnancy, and provide important significance for improving production efficiency. At the same time, EPF can also track the development of the fetus in pregnancy and identify the effect of embryo transfer. Objective: to prepare recombinant protein and monoclonal antibody of sheep early pregnancy factor by molecular biological and cell fusion technique, and to lay a foundation for further development of early pregnancy factor kit. Methods: the expression strain pGEX4t-EPF-BL21DE) was induced by IPTG and the conditions of IPTG were optimized. The expressed recombinant protein of sheep early pregnancy factor EPF-GST was purified by affinity chromatography and identified by SDS-PAGE. The purified EPF recombinant protein was used as antigen to immunize Balb/c mice for 3 times with the interval of 15 days. The titer of mice was detected by indirect ELISA method. The spleen cells and myeloma cells were fused and the hybridoma cells were screened by ELISA method to screen out the positive hybridoma cells and cell lines. The selected cell lines were injected into the abdominal cavity of mice by limited dilution method. The ascites antibody was purified by G-sepharose column affinity chromatography. The purity and specificity of the purified antibody were analyzed by SDS PAGE and Western blot. Results: at 37 鈩，

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