| 摘要: |
| 【目的】利用基因重组技术将草兔卵透明带3基因(ZP3)构建到真核表达载体上，并在RK 13细胞中表达ZP3蛋白，为后期的免疫不育研究提供方便。【方法】提取草兔卵巢总RNA，采用RT PCR方法克隆ZP3基因，将其与pEGFP-N1真核表达载体连接，构建pEGFP-N1-ZP3表达载体，并将载体转入RK-13细胞中进行ZP3表达。利用倒置荧光显微镜、RT-PCR和Western Bolt方法对重组ZP3蛋白表达情况进行观测。【结果】RT-PCR获得长为1 260 bp的草兔ZP3基因；成功构建pEGFP-N1-ZP3重组质粒，将其转染RK-13细胞后表达出73 ku的重组ZP3蛋白，RT-PCR验证和Western Bolt结果与ZP3基因的理论长度一致。【结论】首次构建了草兔ZP3基因真核表达载体并成功在真核细胞中表达重组ZP3蛋白。 |
| 关键词: 草兔 卵透明带3基因 免疫不育 pEGFP-N1 真核表达 |
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| 基金项目:国家林业公益性行业专项（201404405）；西北农林科技大学基本科研业务费项目（QN2012036） |
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| Construction and expression of eukaryotic expression vector of ZP3 gene from Lepus capensis (brown hare) |
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WU Jing-long,SUI Dan-dan,ZHANG Dong-hui,et al
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| Abstract: |
| 【Objective】The eukaryotic expression vector of ZP3 gene from Lepus capensis (brown hare) was constructed by recombinant-DNA technique and expressed in RK-13 cells to improve the study of immunocontraception.【Method】The ovarian RNA was purified from rabbit (Lepus capensis) ovaries.The full-length of ZP3 gene cloned with RT-PCR was connected with the pEGFP-N1 vector to form pEGFP-N1-ZP3 expression vector before being expressed in RK-13 cells.The expression of ZP3 was detected with RT-PCR,Western Blot analysis and fluorescence microscopy.【Result】The full-length of ZP3 gene was 1 260 bp,and the recombinant plasmid pEGFP-N1-ZP3 was successfully constructed.The recombinant ZP3 obtained from RK-13 cells was 73 ku.Western Blot and RT-PCR further proved that the pEGFP-N1-ZP3 was expressed in vitro.【Conclusion】This is the first study that successfully cloned the recombinant ZP3 into eukaryotic expression vector and expressed it in RK-13 cells. |
| Key words: Lepus capensis (brown hare) zona pellucida 3 (ZP3) gene immunocontraception pEGFP-N1 eukaryotic expression |
