猪卵透明带-3β截短型蛋白在原核系统中的表达研究

Study on Expression of Truncated Porcine Zona Pellucida 3β in E.coli

目的:制备具有猪卵透明带-3β抗原活性的截短型pZP3β蛋白(tunc-pZP3β),以便对该蛋白的抗原表位区域作深入研究。方法:采用PCR方法扩增原cDNA中编码第130个氨基酸到第290个氨基酸的DNA序列。获得的tunc-pZP3β基因片段通过引入其两端的NdeⅠ和BamHⅠ酶切位点被定向插入pET-3c质粒T7强启动子下游的多克隆区,筛选出重组子,经DNA测序验证正确后,转化表达菌株BL21(DE3)pLysS,再用IPTG对重组菌体进行诱导。结果:SDS-PAGE分析表明tunc-pZP3β在大肠杆菌系统中高水平表达,且在WesternBlot免疫印迹中能被特异性抗体识别。结论:对适当的抗原表位序列进行保留而截去两端一定的氨基酸序列,所获得的截短型蛋白能够实现在大肠杆菌中高效表达,这有利于深入研究该蛋白核心区域的功能。

Objective: To obtain the recombinant truncated porcine zona pellucida protein 3β (tunc- pZP3β) for further research in its function. Methods: By using PCR , the nucleotides sequence region from 130 to 290 codons of pZP3β entire coding cDNA fragment was obtained. Such sequence was cloned into pET-3c vector. After being identified, recon was transformed into the E.coli BL21 (DE3) pLysS and then induced by IPTG. Results: The recombinant tunc-pZP3β was expressed in E.coli up to 30% of total cellular protein, and was made sure by Western blot analysis. Conclusion: Keeping the epitopes domains, the new truncated protein can meet its high expression in E. coli. This truncated protein could benefit further investigation for its immunogenicity and the development of antigen preparation.