人巨细胞病毒pUL76蛋白非保守C端与蛋白聚集体形成的研究

Unconserved C terminal of pUL76 in human ctomegalovirus elicits aggresome formation

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p UL76引起核内蛋白聚集体形成的决定序列。方法根据Gen Bank中HCMV AD169(FJ527563.1)株基因序列设计分别用于扩增p UL76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(p EGFP-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(Hep G-2),经逆转录(RTPCR)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 p EGFP-N1空载体和p UL76保守N端均不能引起核内蛋白聚集体的形成,而p UL76及其非保守C端均能够引起核内蛋白聚集体的形成。结论 p UL76非保守C端是其引起核内蛋白聚集体形成所必须的。

Objective To define the nuclear agrresome formation is determinated by which part of the UL76 of 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.