CAJ | 학술논문

（目的）构建猪流行性腹泻病毒（Porcine epidemic diarrhea virus，PEDV）E基因真核表达载体pCI—E，为探讨E基因的功能奠定基础。（方法）根据GenBank上已发表的PEDVCV777株序列，利用Primer5．0设计1对两端含限制性核酸内切酶EcoRI和SalI酶切位点的特异引物，用反转录聚合酶链式反应（RT—PCR）扩增出完整的231bp的目的基因，将目的基因与pMD19-T载体连接转化到大肠杆菌DH5a中，用测序、PCR、双酶切鉴定阳性克隆体。然后将纯化后的E基因插入表达载体（pCI—neo）上，并经双酶切、测序鉴定。（结果）成功构建了PEDV的pCI—E重组真核表达载体。（结论）pCI—E重组真核表达载体的构建为进一步探讨PEDVE基因的功能和分子生物学特性及PED防御新途径提供依据。
（목적）구건저류행성복사병독（Porcine epidemic diarrhea virus，PEDV）E기인진핵표체재체pCI—E，위탐토E기인적공능전정기출。（방법）근거GenBank상이발표적PEDVCV777주서렬，이용Primer5．0설계1대량단함한제성핵산내절매EcoRI화SalI매절위점적특이인물，용반전록취합매련식반응（RT—PCR）확증출완정적231bp적목적기인，장목적기인여pMD19-T재체련접전화도대장간균DH5a중，용측서、PCR、쌍매절감정양성극륭체。연후장순화후적E기인삽입표체재체（pCI—neo）상，병경쌍매절、측서감정。（결과）성공구건료PEDV적pCI—E중조진핵표체재체。（결론）pCI—E중조진핵표체재체적구건위진일보탐토PEDVE기인적공능화분자생물학특성급PED방어신도경제공의거。
This study aimed to construct an eukaryotic expression vector of Porcine epidemic diarrhea virus E gene. Based on the published nucleotide sequence of PEDV in Genbank, a pair of specific primers of PEDV was designed by the Primer 5.0 software. To identify and clone the recombinant plasmid, we designed two restriction enzyme sites EcoR I and Sal I in the up- per and down primer, respectively. Following the mixture of the primers, template and the mix reaction system, the complete E gene of PEDV was amplified by RT--PCR. The fragment of the purpose gene was 231bp in length. The purpose gene was connected with pMD19-T vector, named pMD-E, transformed into E. coli DH5a. The positive colonies were confirmed by re- striction enzyme digestion, PCR and sequencing. A recombinant plasmid, named pCI-E was constructed by inserting the E gene into the expression vector pCI-neo, verified by restriction enzyme digestion and sequencing. The recombinant eukaryotic ex- pression vector of the PEDV E gene has been constructed successfully, which provided an experimental basis for the further study of the E gene functions and the PED defense.