| 摘要: |
| [目的]构建猪瘟病毒囊膜蛋白(E2 protein)基因的真核表达载体,并将其在猪脐静脉血管内皮细胞中进行表达.[方法]采用 PCR 技术扩增出E2蛋白全长基因(E2qc)序列和去除跨膜基因(E2sh)序列,并将其克隆入真核表达载体pSec Tag2(A)中,构建pSec Tag-E2qc和pSec Tag-E2sh表达载体,分别进行PCR 、双酶切及测序鉴定.用脂质体法将阳性克隆瞬时转染猪脐静脉血管内皮细胞,对转染细胞进行RT-PCR检测目的基因的转录情况,同时对转染细胞及细胞上清液进行SDS-PAGE和Western-blot分析,以检测目的蛋白的表达情况.[结果]成功克隆了E2蛋白基因全长序列1 119 bp和去除E2蛋白跨膜基因序列999 bp的目的基因.构建的表达载体经PCR、双酶切法及测序鉴定均无误.转染后细胞的RT-PCR结果显示,目的基因被成功转录.转染后细胞及细胞上清液的SDS-PAGE和Western-blot结果显示,目的基因被成功表达.[结论]成功构建了猪瘟病毒E2蛋白的真核表达载体,转染猪脐静脉血管内皮细胞后,分泌表达了猪瘟病毒 E2 重组蛋白. |
| 关键词: 猪瘟病毒囊膜蛋白 猪脐静脉血管内皮细胞 分泌型真核表达载体 真核表达 |
| DOI： |
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| 基金项目:国家高技术研究发展计划(863计划) |
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| Construction of eukaryotic expression vector of CSFV E2 genes and its secretory expression |
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| Abstract: |
| 【Objective】 CSFV envelope protein genes were subcloned into a eukaryotic expression vetor which could make the protein secret into medium,and then swine umbilical vein endothelical cell（SUVECs）was used to express this protein.【Method】 CSFV E2 full-length genes (E2qc)and E2 genes without transmembrane sequence(E2sh) were amplified from plasmid pMD19T-E2 by PCR,then the two genes were subcloned into pSec Tag 2（A）;PCR digestion and sequencing were used to identify positive plasmid;the identified recombined plasmid was transiently transfected into SUVECs by Lipofectamine 2000 and expression products in SUVECs were determined by RT-PCR、SDS-PAGE and Western blot.【Result】 The fragments of E2 full-length genes and E2 genes without transmembrane sequence were examined by 10 g/L agarose electrophoresis consistent with predicted size.PCR digestion and sequencing showed that the recombinant plasmids were accurate；E2 protein expressing in SUVECs was confirmed by RT-PCR and Western-blot.The results showed that the E2 protein was secteted into the medium.【Conclusion】 Recombinant plasmid pSec Tag-E2 containing CSFV envelope protein genes was successfully constructed,and E2 protein made secretory expression in SUVECs. |
| Key words: classical swine fever virus envelope protein SUVECs recombinant plasmid secretory expression |
