家兔精子膜蛋白rSP10在大肠杆菌中的高效表达及其抗血清的制备  

High Expression of Rabbit Sperm Membrane Protein rSP10 in Escherichia coli and Preparation of its Specific Antisera

作　　者：刘建喜[1] 林爱星[1] 杨毅[2] 侯健[1] 胡宏宇[1] 陈永福[1] 

机构地区：[1]中国农业大学农业生物技术国家重点实验室,北京100094 [2]中国农业大学生物学院,北京100094

出　　处：《生物工程学报》2001年第3期314-317,共4页Chinese Journal of Biotechnology

摘　　要：为研究家兔精子膜蛋白rSP10在精子膜上的定位及其免疫原性 ,将不含编码信号肽序列的rsp10基因插入表达载体pET30a中 ,N端具有His6 肽的融合蛋白re rSP10得到高效表达 ,表达产物达细菌总蛋白的 6 7%。经DEAE柱层析纯化表达产物 ,re rSP10 ,产量约为 5 0mg mL培养物。Western实验结果表明 ,精子膜蛋白多克隆抗体能识别re rsp10 ,说明大肠杆菌表达的re rSP10具有免疫原性。用纯化的re rSP10免疫雌性兔 ,得到re rSP10专一性多克隆抗血清。将re rSP10专一性多克隆抗血清加入获能精子中 ,发现SP10专一性多克隆抗血清严重影响获能精子的运动并且表现为剂量依赖性 。To study the position and immunogenicity of rSP10,the rabbit rsp10 gene which did not include sequences coding for signal peptides was inserted into expression vector pET30a.An in\|frame fusion protein was made such that a His 6 stretch was produced at the N terminus of re\|rSP10.High expression was obtained,the amount of re\|rSP10 up to 67% in the total bacterial protein.The re\|rSP10 was purified by DEAE chromatography and the yield of purified re\|rSP10 was approximately 50μg/mL of culture.Western blotting analysis of re\|rSP10 with rabbit polyclonal sera raised against rabbit sperm membrane protein showed that the synthesized antigen possessed immunogenicity of rSP10.Specific antisera against re\|rSP10 was induced using purified re\|rSP10 as an antigen.The motility of capacitated sperms were affected but no aggregation was observed.

关 键 词：家兔 精子膜蛋白 rSP10 表达 抗血清 免疫避孕 

分 类 号：Q51[生物学—生物化学]