昆虫细胞杆状病毒表达系统表达重组人类ZP3蛋白的研究

目的:探讨利用重组表达系统制备重组人类ZP3(h ZP3)蛋白的方法和技术难点,为h ZP3蛋白的制备和应用打下技术基础。方法:构建h ZP3的载体质粒p FASTBAC HTa-h ZP3,在大肠杆菌宿主细胞中利用杆状病毒Bac-to-Bac系统同源重组构建重组的杆状病毒穿梭载体Bacmid,用重组的Bacmid转染Sf9细胞制备重组杆状病毒,分别表达带HIS标签的h ZP3全长蛋白(氨基酸1~424)和截短的h ZP3蛋白(氨基酸23~348)。摸索重组h ZP3蛋白的表达时间和纯化制备方法。结果:成功制备了表达重组人h ZP3全长蛋白和截短体蛋白的Bacmid和杆状病毒颗粒,Western印迹检测表明,在Sf9细胞中较好的表达时间段为感染后72~96 h。重组表达的h ZP3蛋白可以部分被钴柱亲和纯化,大量的重组h ZP3蛋白不能结合到亲和介质而流穿,但确切的原因还未知。纯化得到的h ZP3经荧光素Dylight Dye488标记,能与人精子结合。结论:利用昆虫细胞杆状病毒表达系统表达h ZP3蛋白是可行的,可以进一步优化,但h ZP3蛋白的有效富集和纯化方法是最大的瓶颈,还需要进一步摸索。

Expression of recombinant human ZP3 protein using the baculovirus expression system

Objective： To investigate the methods and solve the technical bottlenecks in the preparation of recombinant human protein h ZP3 using the baculovirus expression system and pave the technical ground for the production and application of recombinant hZP3. Methods： The recombinant vector pFASTBAC HTa-hZP3 was constructed and transferred to competent E. coli cells carrying bacmid to produce recombinant bacmid by homologous recombination. Sf9 cells were transfected with the recombinant bacmid to produce recombinant baculovirus. Full-length recombinant h ZP3（ amino acids 1-424） and truncated recombinant hZP3（ amino acids 23-348） were expressed in the sf9 cells by infection with the recombinant baculovirus. The expression time of hZP3 was determined by Western blot and its purification was explored. Results： The recombinant bacmid and baculovirus were successfully constructed for expressing both the full-length and truncated hZP3. The maximal expression of recombinant hZP3 in the sf9 cells was achieved at 72-96 hours after baculovirus infection. Some of the recombinant h ZP3 with His-tag could bind affinity matrix and got purified but most of the solubilized h ZP3 passed through and the reasons remained unknown. Purified recombinant h ZP3 labeled with Dylight Dye488 was able to bind human sperm. Conclusion： It is feasible to express recombinant h ZP3 in insect cells using the baculovirus system though the yield of hZP3 needs to be optimized. The methods for efficient enrichment and purification of recombinant hZP3 require further exploration.