CAJ | 학술논문

目的：构建登革2型病毒（DENV2）NS1基因的真核表达载体，为筛选与NS1相互作用的蛋白以及研究机体抵抗登革病毒感染的作用机制奠定基础。方法以DENV2感染THP1细胞的cDNA为模板，采用RT-PCR法扩增具有Flag标签的NS1全长基因，并将其克隆至pSG5质粒中，构建pSG5-NS1-Flag真核表达载体。筛选阳性克隆，分别进行酶切及测序鉴定。采用脂质体转染法将真核表达载体分别转染293T细胞和A549细胞，Western blot-ting法检测细胞NS1蛋白表达。结果扩增后具有Flag标签的NS1全长基因片段大小为1089 bp，与预期目的片段大小相符。载体和目的基因NS1都含1个ECORⅠ位点，单酶切片段大小分别为463、4722 bp，NS1扩增片段和pSG5质粒的双酶切片段大小分别为1089、4100 bp；6个阳性克隆中，5、6号克隆酶切片段符合预期大小，并经序列鉴定证实。293T细胞和A549细胞转染空载体后，NS1融合蛋白表达极低，转染真核表达载体后均有NS1融合蛋白表达。结论本研究成功构建了pSG5-NS1-Flag真核表达载体，为与NS1相互作用的免疫调控蛋白、NS1蛋白翻译后修饰以及机体免疫系统抵御登革病毒感染机制的相关研究奠定了基础。
목적：구건등혁2형병독（DENV2）NS1기인적진핵표체재체，위사선여NS1상호작용적단백이급연구궤체저항등혁병독감염적작용궤제전정기출。방법이DENV2감염THP1세포적cDNA위모판，채용RT-PCR법확증구유Flag표첨적NS1전장기인，병장기극륭지pSG5질립중，구건pSG5-NS1-Flag진핵표체재체。사선양성극륭，분별진행매절급측서감정。채용지질체전염법장진핵표체재체분별전염293T세포화A549세포，Western blot-ting법검측세포NS1단백표체。결과확증후구유Flag표첨적NS1전장기인편단대소위1089 bp，여예기목적편단대소상부。재체화목적기인NS1도함1개ECORⅠ위점，단매절편단대소분별위463、4722 bp，NS1확증편단화pSG5질립적쌍매절편단대소분별위1089、4100 bp；6개양성극륭중，5、6호극륭매절편단부합예기대소，병경서렬감정증실。293T세포화A549세포전염공재체후，NS1융합단백표체겁저，전염진핵표체재체후균유NS1융합단백표체。결론본연구성공구건료pSG5-NS1-Flag진핵표체재체，위여NS1상호작용적면역조공단백、NS1단백번역후수식이급궤체면역계통저어등혁병독감염궤제적상관연구전정료기출。
Objective To construct a eukaryotic expression vector of dengue virus serotype 2( DENV2) NS1 gene, and to lay the foundation for screening the protein which was interacting with NS 1 and for the mechanism of being against the dengue virus infection .Methods NS1-Flag sequence was amplified from the DENV 2-infected THP1 genomic DNA by RT-PCR and cloned into pSG5 plasmid to construct the pSG5-NS1-Flag eukaryotic expression vector .Positive clones were screened and then were identified by the enzyme digestion and sequencing .The eukaryotic expression vector was transfected into 293T cells and A549 cells by lipofection transfection .The expression of NS1 protein was detected by Western blotting . Results The full-length gene fragment size of NS 1after amplification which had the Flag label was 1 089 bp, and was con-sistent with the expected one .The carrier and NS1 gene both contained 1 of ECORⅠsite, the single enzyme fragment sizes were 463 and 4 722 bp, respectively;the amplified fragment size of NS 1 and the double enzyme fragment size of pSG 5 plas-mid were 1 089 and 4 100 bp, respectively .During the six positive clones , the target bands of clone 5 and clone 6 were consistent with the expected results and were identified by sequence identification .The NS1 fusion protein expression was extremely low after 293T cells and A549 cells were transfected by empty vector , after the transfection of eukaryotic expres-sion vector , there was the NS1 protein expression .Conclusion The pSG5-NS1-Flag eukaryotic expression vector is suc-cessfully constructed , which paves the way for studying interaction between NS 1 and immunomodulatory protein , post-trans-lational modification and the mechanism of the body′s immune system against dengue virus infection .