【目的】制备番鸭丝裂原活化蛋白激酶1(mitogen-activated protein kinase,MAPK1)蛋白多克隆抗体,鉴定其特异性与适用性,并探究MAPK1蛋白在卵泡颗粒细胞及卵泡中的表达定位,为研究其在番鸭卵泡发育与生殖调控中的功能提供试验工具。【...【目的】制备番鸭丝裂原活化蛋白激酶1(mitogen-activated protein kinase,MAPK1)蛋白多克隆抗体,鉴定其特异性与适用性,并探究MAPK1蛋白在卵泡颗粒细胞及卵泡中的表达定位,为研究其在番鸭卵泡发育与生殖调控中的功能提供试验工具。【方法】以番鸭卵巢组织cDNA为模板,通过PCR扩增MAPK1基因并测序,运用生物信息学工具分析MAPK1蛋白理化性质及主要抗原表位,筛选免疫原区段。对免疫原序列进行大肠杆菌密码子优化并人工合成,经同源重组克隆至原核表达载体pET-30a(+),构建重组表达载体pET30a-MAPK1并转化大肠杆菌BL21(DE3)感受态细胞。在HB-PET自诱导培养基中表达、纯化MAPK1重组蛋白,将纯化蛋白与QuickAntibody-Rabbit8W佐剂混合后免疫新西兰白兔制备多克隆抗体。采用ELISA、Western blotting、细胞免疫荧光(CIF)、组织免疫荧光(TIF)和免疫组化(IHC)等方法检测多克隆抗体的效价、特异性与适用性,并以颗粒细胞标志物促卵泡激素受体(FSHR)为参照分析MAPK1表达定位。【结果】成功克隆了番鸭MAPK1基因,大小约1107 bp,编码368个氨基酸。MAPK1蛋白理论分子质量约42 ku。生物信息学预测显示,MAPK1蛋白不含信号肽和跨膜结构;第50—100、200—300和250—350位氨基酸区段亲水性良好,第30—90和200—300氨基酸区段抗原指数较高,第30—70、120—150、200—260及320—360位氨基酸区段的氨基酸表面暴露概率较大。MAPK1蛋白二级结构以α-螺旋为主,包含典型激酶保守结构,活化环呈无规则卷曲状态,主要定位于细胞质和细胞核。综合蛋白特性,最终筛选了第4—364位氨基酸作为候选免疫原区段。成功构建原核重组表达载体pET30-MAPK1,MAPK1重组蛋白以可溶性形式表达,分子质量约48 ku。成功制备了兔抗鸭MAPK1蛋白多克隆抗体,ELISA结果显示该抗体效价达1∶102400;Western blotting、CIF、TIF和IHC结果显示,制备的多克隆抗体可特异识别MAPK1重组蛋白及番鸭卵泡颗粒细胞和卵泡中的内源性MAPK1蛋白。在颗粒细胞中,MAPK1主要定位于卵泡颗粒细胞的细胞质和细胞核;在卵泡中,MAPK1主要分布于卵泡颗粒细胞层,表达定位与FSHR基本一致。【结论】试验成功制备了番鸭MAPK1多克隆抗体,该抗体特异性良好,适用于番鸭卵泡相关细胞与组织样本中MAPK1的免疫学检测。研究结果为MAPK1在卵泡发育及生殖调控中的功能研究提供了技术支撑。展开更多
[Objective] This study aimed to clone the E. coil NrfA gene and construct the pET-28a （＋）-NrfA prokaryotic expression vector for preparation of polyclonal anti- body against E. coil NrfA. [Method] E. coil NrfA gene...[Objective] This study aimed to clone the E. coil NrfA gene and construct the pET-28a （＋）-NrfA prokaryotic expression vector for preparation of polyclonal anti- body against E. coil NrfA. [Method] E. coil NrfA gene was cloned from the E. coli genome DNA by PCR and inserted into the vector pET-28a（＋） to construct prokary- otic expression vector pET-28a （＋）-NrfA. E. coil NrfA protein was expressed by IPTG induction and purified. Polyclonal antibody against NrfA protein was prepared by im- munizing rabbit with routine method. The specificity and titer of polyclonal antibody was confirmed by ELISA and Western Blotting. [Result] The constructed prokaryotic expression vector pET-28a（＋）-NrfA was induced by IPTG, the recombinant NrfA pro- tein could be expressed effectively. The titer of rabbit anti-NrfA polyclonal antibody obtained by immunization and purification was about 1：204 900. Western Blotting anal- ysis indicated that the obtained polyclonal antibody against E. coil NrfA protein had high titer and high specificity. [Conclusion] E. coil NrfA gene was cloned and the prokaryotic expression vector pET-28a （＋）-NrfA was constructed successfully, poly- clonal antibody with high titer and high specificity was prepared, which laid the foun- dation for the study of NrfA in different strains of bacteria.展开更多
文摘[Objective] This study aimed to clone the E. coil NrfA gene and construct the pET-28a （＋）-NrfA prokaryotic expression vector for preparation of polyclonal anti- body against E. coil NrfA. [Method] E. coil NrfA gene was cloned from the E. coli genome DNA by PCR and inserted into the vector pET-28a（＋） to construct prokary- otic expression vector pET-28a （＋）-NrfA. E. coil NrfA protein was expressed by IPTG induction and purified. Polyclonal antibody against NrfA protein was prepared by im- munizing rabbit with routine method. The specificity and titer of polyclonal antibody was confirmed by ELISA and Western Blotting. [Result] The constructed prokaryotic expression vector pET-28a（＋）-NrfA was induced by IPTG, the recombinant NrfA pro- tein could be expressed effectively. The titer of rabbit anti-NrfA polyclonal antibody obtained by immunization and purification was about 1：204 900. Western Blotting anal- ysis indicated that the obtained polyclonal antibody against E. coil NrfA protein had high titer and high specificity. [Conclusion] E. coil NrfA gene was cloned and the prokaryotic expression vector pET-28a （＋）-NrfA was constructed successfully, poly- clonal antibody with high titer and high specificity was prepared, which laid the foun- dation for the study of NrfA in different strains of bacteria.
