人巨细胞病毒临床病毒株UL148基因的体外表达及功能位点预测  

In vitro expression of human cytomegalovirus UL148 RNA and prediction of its functional sites

作　　者：胡兢晶[1] 伍苑宾[1] 谭琪琪 苏海浩[1] 丁俊彩[1] 郭媛媛[1] 谢斌华[1] 蔡丽君[1] 郭梦杰[1] 王波[1] Hu Jingjing;Wu Yuanbin;Tan Qiqi;Su Haihao;Ding Juncai;Guo Yuanyuan;Xie Binhua;Cai Lijun;Guo Mengjie;Wang Bo(Department of Pediatrics, Guangdong Women and Children＇s Hospital, Guangzhou 511442, China)

机构地区：[1]广东省妇幼保健院儿科,广州511442

出　　处：《中华微生物学和免疫学杂志》2018年第2期94-97,共4页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（81070516）;广东省自然科学基金（2015A030313726,2016A030313788,9151008901000085）;广东省医学科研基金（A2015133,A2009078）;广东省妇幼保健院国家自然科学基金院内培育项目（YN2017G06）

摘　　要：目的 体外获得人巨细胞病毒（human cytomegalovirus, HCMV）临床病毒株 UL148 RNA及探讨其基因功能.方法 将感染HCMV的新生儿尿液接种人胚肺细胞,分离HCMV临床病毒株,并经多重PCR鉴定.扩增UL148基因,经双酶切后克隆入pGEM-T-Easy质粒,构建重组质粒,并置于T7启动子下游,经重组质粒、PCR产物、双酶切产物电泳鉴定及序列测定.克隆成功的质粒以32P标记,进行体外转录RNA.根据UL148序列特征应用生物信息学分析其翻译后修饰位点.结果 成功在体外获得HCMV临床病毒株,经电泳及序列测定证实重组质粒构建成功,在T7 RNA聚合酶作用下,体外获得UL148 RNA.翻译后修饰位点显示UL148基因存在1个与细胞黏附相关的特征序列,1个豆荚外源凝集素β-链标记位点、2个N-豆蔻酰化位点,1个酪氨酸激酶Ⅱ磷酸化位点,7个蛋白激酶C磷酸化位点,1个cAMP/cGMP-依赖性蛋白激酶磷酸化位点和2个N-糖基化位点,1个跨膜区域.结论 UL148基因编码产物可能为一个病毒黏附分子,参与宿主细胞的信号转导作用.Objective To express UL148 RNA of human cytomegalovirus （HCMV） clinical strains in vitro and to study its functions. Methods Urine of a newborn with HCMV infection was inocula-ted into human embryo lung cells. HCMV clinical strain was isolated and identified by multiplex PCR. UL148 gene was amplified and cloned into pGEM-T-Easy plasmid after double enzyme digestion. A recombi-nant plasmid was constructed and located at the downstream of the T7 promoter. The recombinant plasmid was identified by electrophoresis of the recombinant plasmid,PCR product and double enzyme product. Se-quencing analysis was used for final confirmation. UL148 was transcribed into RNA by 32P labeling. Post-translational modification sites were analyzed by bioinformatics method based on UL148 sequence characteris-tics. Results The clinical strain of HCMV was obtained in vitro. Electrophoresis and sequencing analysis confirmed the successful construction of the recombinant plasmid. UL148 RNA was transcribed in vitro by T7RNA polymerase. Post-translational modification sites showed that UL148 gene contained one cell adhe-sion sequence, one legume lectins beta-chain signature, two N-myristoylation sites, one casein kinase Ⅱphosphorylation site,seven protein kinase C phosphorylation sitse, one cAMP/cGMP-dependent protein ki-nase phosphorylation site, two N-glycosylation sites and one transmembrane region. Conclusion UL148 gene might encode a viral adhesion molecule involving in the signal transduction in host cells.

关 键 词：人巨细胞病毒 临床病毒株 UL148 信号转导 

分 类 号：R373[医药卫生—病原生物学]