發(fā)布時間：2018-04-27 23:49

  本文選題：綿羊 + 早孕因子　； 參考：《河北農業(yè)大學》2015年碩士論文

【摘要】：早孕因子(Early pregnancy factor,EPF)是母體受精后從血清中檢測出最早與妊娠相關的蛋白,是與妊娠相關的免疫抑制因子,被認為是最早能確認妊娠的生化指標之一。在養(yǎng)殖生產中,利用早孕因子的特異性可以及時進行綿羊的妊娠診斷,減少其空懷發(fā)生,為提高生產效率提供重要意義。同時,EPF還可以跟蹤妊娠母體中胎兒的發(fā)育情況,鑒定胚胎移植效果。目的:應用分子生物和細胞融合技術制備了綿羊早孕因子重組蛋白和單克隆抗體,為進一步研制早孕因子試劑盒奠定基礎。方法:對表達菌p GEX4t-EPF-BL21(DE)進行IPTG誘導劑,對誘導劑IPTG進行條件優(yōu)化。將表達出的綿羊早孕因子重組蛋白(EPF-GST),通過親和吸附層析柱對蛋白進行純化,并用SDS-PAGE進行鑒定。以純化后的EPF重組蛋白為抗原免疫Balb/c小鼠,免疫3次,每次免疫時間間隔為15天,后采用間接ELISA方法檢測小鼠效價,取效價合格小鼠(N/P"g2)的脾細胞,將脾細胞和骨髓瘤細胞進行細胞融合,對雜交瘤細胞進行ELISA方法進行篩選,篩選出陽性雜交瘤細胞和細胞株。經有限稀釋法將篩選的細胞株注入小鼠腹腔,用G-sepharose柱親合層析法對小鼠腹水型抗體進行純化,應用SDS PAGE和Western blot分析抗體純化后的純度及特異性。結果:在37℃下,表達菌最佳優(yōu)化條件為:IPTG濃度0.5mmol/m L,誘導時間4h,在此誘導條件下表達菌能最大量表達綿羊EPF重組蛋白,重組蛋白分子量大小為26KD(其中早孕因子蛋白為10KD,標簽蛋白為16KD),且該表達重組蛋白以可溶性形式存在。用純化后的重組蛋白免疫小鼠,小鼠血清效價可達1:16000。取小鼠的脾細胞與骨髓瘤細胞融合,得到了1株抗綿羊EPF單克隆抗體的細胞株B4D5,效價為1:1000。腹水型抗體經G-sepharose柱親合層析,SDS PAGE和Western blot檢測,得到純度較高的單克隆抗體,且與綿羊EPF具有一定的特異性。結論:從表達菌中制備出了綿羊EPF重組蛋白(EPF-GST),以重組蛋白免疫小鼠,得到了綿羊早孕因子單克隆抗體。
[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，

本文編號：1812872