小鼠ZP2肽的基因克隆和体外表达.pdfVIP

中文摘要 中文摘要 目的：克隆小鼠卵透明带 ZP2 肽段基因序列，并利用原核表达系统表达重组 ZP2 肽 段，以便进一步研究其免疫活性和生理功能。 方法：以 RT-PCR 法扩增小鼠卵透明带 ZP2 肽段的 DNA 序列并插入到 pET 载体构 建重组质粒。经 PCR 、酶切和测序鉴定后转化工程菌株 Rosetta 进行诱导表达。用 SDS和 Western blotting 分析鉴定表达产物。重组蛋白经 SDS分离后，将目 的蛋白条带切下，研磨碎，与弗式佐剂乳化后免疫新西兰雄兔，制备抗重组mZP2 特异抗 原的抗血清。ELISA 法检测抗血清的抗体效价。用Western blotting 检测抗血清与重组mZP2 抗原的特异性反应。免疫组化检测抗血清与小鼠卵巢组织的特异性免疫反应。 结果： PCR 、酶切和测序的结果显示克隆了mZP2 肽段的 DNA 序列。构建了 mZP2 原核表达质粒载体。SDS和 Western blotting 结果显示，重组质粒转化的 Rosetta 工 程菌株表达出特异的重组蛋白。重组蛋白的分子大小约 35kD 左右。ELISA 和 Western Blotting 结果说明用重组蛋白免疫新西兰雄兔诱发产生了抗血清。免疫组化实验证明抗血 清与小鼠卵巢透明带发生了特异性反应。 结论：克隆的mZP2 肽基因在原核表达系统中获得表达，重组 mZP2 蛋白具有很好的 免疫原性。 关键词：小鼠卵透明带mZP2 ， 基因克隆，基因表达 I 英文摘要 Abstract Objective: To clone the gene of mouse ZP2 peptide and express the recombinant proteins in the prokaryotic expression system. Method ：The total RNA were extracted from mouse ovary, the cDNA sequence of mZP2 was reversely transcribed by RT-PCR method. then the mZP2 DNA fragment was inserted in the MCS of the prokaryotic expression vector pET and confirmed by PCR and restriction enzyme digestion. the recombinant protein was expressed in Rosetta with IPTG induction. The recombinant proteins were separated by SDSand identified by Western blotting. To obtain rabbit anti-mZP2 antiserum, the recombinant mZP2 was isolated by SDSand the gel of the specific band were cut and ground fine. The gel particles were emulsified with freund，s adjuvant and injected intramuscularly in the New Zealand white rabbits. The antibody response of the antiserum were detected by ELISA ，the specific binding of an