CAJ | 학술논문

目的：通过分别构建人巨细胞病毒( HCMV) UL76基因编码蛋白的全长以及保守N端和非保守C端的真核表达质粒，探讨 pUL76引起核内蛋白聚集体形成的决定序列。方法根据GenBank中HCMV AD169(FJ527563.1)株基因序列设计分别用于扩增pUL76全长以及保守N端和非保守C端的引物，将3段序列分别构建至增强型绿色荧光蛋白表达载体(pEGFP-N1)中，经双酶切和测序验证重组质粒的正确构建。空载体和3种重组质粒分别瞬时转染人胚肺成纤维细胞( HELF )和人肝癌细胞( HepG-2)，经逆转录( RT-PCR)和Western blot法验证各段基因的正确表达，在荧光显微镜下观察转染不同重组质粒后细胞核内蛋白聚集体形成的状态。结果 pEGFP-N1空载体和pUL76保守N端均不能引起核内蛋白聚集体的形成，而pUL76及其非保守C端均能够引起核内蛋白聚集体的形成。结论 pUL76非保守C端是其引起核内蛋白聚集体形成所必须的。
목적：통과분별구건인거세포병독( HCMV) UL76기인편마단백적전장이급보수N단화비보수C단적진핵표체질립，탐토 pUL76인기핵내단백취집체형성적결정서렬。방법근거GenBank중HCMV AD169(FJ527563.1)주기인서렬설계분별용우확증pUL76전장이급보수N단화비보수C단적인물，장3단서렬분별구건지증강형록색형광단백표체재체(pEGFP-N1)중，경쌍매절화측서험증중조질립적정학구건。공재체화3충중조질립분별순시전염인배폐성섬유세포( HELF )화인간암세포( HepG-2)，경역전록( RT-PCR)화Western blot법험증각단기인적정학표체，재형광현미경하관찰전염불동중조질립후세포핵내단백취집체형성적상태。결과 pEGFP-N1공재체화pUL76보수N단균불능인기핵내단백취집체적형성，이pUL76급기비보수C단균능구인기핵내단백취집체적형성。결론 pUL76비보수C단시기인기핵내단백취집체형성소필수적。
Objective To define the nuclear agrresome formation is determinated by which part of the UL76 of <br> HCMV. Full-length, conserved N terminal and unconserved C terminal of pUL76 were constructed to eukaryotic ex-pression plasmid pEGFP-N1 . Methods Primers were designed to amplify full-length and different part of pUL76 according to standard sequence of HCMV AD169 which had been submitted to GenBank(FJ527563. 1). These frag-ments were constructed to eukaryotic expression plasmid pEGFP-N1 . The recombinant plasmids were designated pEGFP-UL76,pEGFP-UL76N, pEGFP-UL76C respectively. Double digestion and sequencing were performed to verify the accuracy of recombinant plasmids construction. Empty vector and three recombinant plasmids were transi-ent transfected to HELF and HepG-2 cells respectively. Reverse transcriptation PCR and Western blot were per-formed to detect the RNA and protein expression level respectively. Different nuclear aggresome formations were visualized with an Olympus fluorescence microscopy. Results pEGFP-N1 and pEGFP-UL76N were unable to in-duce nuclear aggresome formation, whereas pEGFP-UL76 and pEGFP-UL76C were able to elicit nuclear aggresome formation. Conclusion The unconserved C terminal of pUL76 is sufficient to induce nuclear aggresome formation.