CAJ | 학술논문

目的构建乙脑病毒prME蛋白与鼠GM-CSF融合基因重组子并建立稳定细胞表达株。方法套式-RT—PCR法从BALB／c鼠脾组织获取GM＋CSF编码基因，用限制性内切酶从含乙脑病毒wME蛋白基因重组子获取prME蛋白基因，定向克隆至同-真核表达载体pcDNA3．1（＋）不同酶切位点，构建重纽子pJME／GM—CSF并经酶切及DNA测序分析。脂质体法将pJME／GM—CSF转染中华仓鼠卵巢（CHO）N胞。Western blot法检测转染的CHO细胞中融合蛋白表达。结果pJME／GM—CSF经BamHI／EcoRI和BamHI／NotI酶切释出的插入子片段（2001bp、2472bp）分别与预期结果相符合。所编码的融合蛋白相对分子量（Mr）为85×103。结论pJME／GM-CSF成功构建，转染的CHO细胞可稳定表达融合蛋白。
목적구건을뇌병독prME단백여서GM-CSF융합기인중조자병건립은정세포표체주。방법투식-RT—PCR법종BALB／c서비조직획취GM＋CSF편마기인，용한제성내절매종함을뇌병독wME단백기인중조자획취prME단백기인，정향극륭지동-진핵표체재체pcDNA3．1（＋）불동매절위점，구건중뉴자pJME／GM—CSF병경매절급DNA측서분석。지질체법장pJME／GM—CSF전염중화창서란소（CHO）N포。Western blot법검측전염적CHO세포중융합단백표체。결과pJME／GM—CSF경BamHI／EcoRI화BamHI／NotI매절석출적삽입자편단（2001bp、2472bp）분별여예기결과상부합。소편마적융합단백상대분자량（Mr）위85×103。결론pJME／GM-CSF성공구건，전염적CHO세포가은정표체융합단백。
Objective Construction and expression of recombinant plasmid fusing encoding prME protein derived from Japanese encephalitis virus （JEV） and GM-CSF of BALB/c mouse. Methods GM-CSF gene was ampli/ied by nested-RT-PCR from BALB/c spleen cells. JEV prME protein gene was eluted by the digestion with restrict/on endonucleases BamH I/EcoR I from pJME piasmid. Genetic fusion of prME protein and GM-CSF are subcloned info pcDNA3.1 （＋） eukaryotic vector, and named as pJME/GM-CSF.The recombinant was confirmed by restriction enzymes digestion and DNA sequencing, and then transfected into China hamster ovary （CHO） cells by Lipofectamine2000. pJME/ GM-CSF expression in CHO cells was examined by Western blot. Results We observed 2001 bp and 2472 bp DNA fragments when pJME/GM-CSF was digested with BamHI/EcoRI and BamHI/NotI respectively as expected. The estimated molecular weight of the fusion protein was 85kD. Conclusion The recombinant pJME/GM-CSF was constructed and transfected into CHO cells successfully with pJME/GMCSF stably expressed.