【作者】 苏海霞；

【导师】 闫永平； 徐德忠； 张景霞； 赵小宁；

【作者基本信息】 第四军医大学 ， 流行病与卫生统计学， 2007， 博士

【摘要】 目的和意义丙型肝炎病毒（HCV）是慢性肝炎和肝癌的主要病原体之一,但对于HCV的复制和致病机制目前还是不甚清楚。宿主细胞中的许多蛋白分子可以通过与HCV RNA基因组的相互作用,调节HCV的翻译、复制和病毒的组装。现有研究发现的HCV RNA结合蛋白,其结合区域主要位于HCV RNA的5’-UTR、3’-UTR和RNA的副链。但是与HCV CORE区（核心区）RNA结合的细胞蛋白却很少有研究报导。本研究拟采用凝胶迁移和紫外交联的实验方法,从人肝癌细胞系HepG2细胞中初步筛选与HCV CORE区RNA结合的细胞蛋白分子,确定其与HCV RNA结合的具体区域,并对RNA结合蛋白进行分离、鉴定。通过研究宿主细胞蛋白与HCV C区RNA的相互作用,将有助于我们对宿主和病毒相互作用的更好理解,从而在细胞蛋白质水平对病毒RNA的翻译、复制和病毒蛋白表达进行调控;并有利于在丙型肝炎的预防和治疗方面提出针对性的措施。方法①采用RT-PCR扩增HCV C区cDNA序列,并构建重组质粒pGEM-HCV;以pGEM-HCV为模板,采用体外转录的方法制备DIG标记和未标记的HCV C-RNA分子;将DIG标记的HCV C-RNA与HepG2细胞蛋白结合,进行凝胶迁移实验;将DIG标记的HCV C-RNA与HepG2细胞蛋白结合,进行紫外交联筛选实验,并采用未标记的HCV C-RNA分子进行竞争性实验;电泳后将RNA/蛋白转移至NC膜,以DIG抗体检测NC膜上的DIG信号,判断是否有细胞蛋白与HCV C-RNA结合。②采用PCR、克隆和体外转录的方法,制备不同长度的HCV C-RNA分子;分别将不同长度的HCV C-RNA分子与细胞蛋白进行结合反应和紫外交联实验,确定HCV C-RNA与细胞蛋白的结合区域;采用抗-DIG和免疫沉淀方法,从RNA和蛋白混合物中,分离与DIG标记的HCV C-RNA分子结合的细胞蛋白;通过SDS-PAGE和NC膜的检测结果的比对,切取目的蛋白条带,进行肽指纹图谱分析鉴定。③提取HepG2细胞总RNA,以RT-PCR方法扩增PGAM-B的全基因片断,与载体pcDNA^TM3.1/V5-HisA连接,构建真核表达质粒pcDNA-PGAM;以PCR方法扩增pGEM-HCV中HCV核心蛋白基因片断,与载体pcDNA3.0连接,构建真核表达质粒pcDNA-HCV;经酶切和测序鉴定后,将真核表达质粒分别转染HepG2细胞;以免疫细胞化学的方法,在细胞爬片上分别检测细胞中PGAM-His蛋白和HCV核心蛋白的瞬时表达情况。结果①从1例HCV感染者血清中扩增得到的503bp HCV cDNA序列,构建了重组质粒pGEM-HCV,并进行了PCR、酶切鉴定和测序鉴定。②所得HCV cDNA序列与已知HCV序列（AB092962.1）比较,属于HCV 1型基因C区,仅有两个核苷酸不同,nt 67（23aaK→E）和nt 177（沉默突变）,其余序列均一致。③通过凝胶迁移实验初步检测到,有HepG2细胞蛋白与HCV C-RNA分子结合。④通过紫外交联实验检测到,有多个HepG2细胞蛋白与HCVC-RNA分子在体外发生了结合;随着结合反应体系中细胞蛋白浓度的增加,HCV C-RNA和蛋白的结合量随之增多;未标记HCV C-RNA对DIG标记的HCV C-RNA与HepG2细胞蛋白的结合有竞争性抑制作用。⑤3个不同长度的HCV C-RNA分子（198nt、306nt和503nt）都可与HepG2细胞蛋白在体外发生结合;其中C-RNA 5’端的C198 RNA与细胞蛋白的结合条带,最为清晰和锐利。⑥经免疫沉淀后的蛋白,在NC膜上可以检测到4条明显的兰紫色RNA结合蛋白带;其中以P30蛋白条带最为清晰,蛋白量最多。⑦从SDS-PAGE胶上切取了P30蛋白条带,经肽指纹图谱分析鉴定,P30蛋白为磷酸甘油酸变位酶1（PGAM-B）。⑧从HepG2细胞中,扩增得到了约为760bp的PGAM-B的cDNA全基因片断;所得PGAM-B基因序列与已知PGAM-B基因序列（J04173）进行比较,完全一致。⑨基因序列和蛋白读码框均正确的真核表达质粒pcDNA-PGAM和pcDNA-HCV,分别转染HepG2细胞后,可在细胞爬片上检测到PGAM-His和HCV核心蛋白的瞬时表达。结论①在HepG2细胞中,有多个细胞蛋白分子可以与HCV核心区RNA在体外发生特异性的结合。②HCV C-RNA与HepG2细胞蛋白的结合区域可能位于HCV C区RNA的5’端。③PGAM-B可以与HCV C-RNA在体外发生特异结合,提示PGAM-B可能通过与HCV核心区RNA的相互作用,参与HCV RNA的翻译和复制。④PGAM-B和HCV核心蛋白可以在HepG2细胞中瞬时表达。更多还原

【Abstract】 Objective and significanceHepatitis C virus (HCV) is one of causative pathogens of chronic hepatitis and liver cancer. However the molecular mechanisms underlying HCV replication and pathogenesis are poorly understood. A number of cellular factors from host cells may be involved in regulating HCV translation, replication and assembly by interacting with HCV RNA genome. These studies on HCV RNA-binding proteins were mainly focused on the 5’-UTR, 3’-UTR and negative strand of HCV RNA genome. But the studies on cellular proteins binding to core region of HCV RNA genome are still few. The objectives of this study were to screen, separate and identify cellular proteins, which could bind to core region of HCV RNA genome. It will help us to know about host cell-virus interactions, to regulate HCV translation and repulication at cellular level and to take measures in prevention and treatment of hepatitis C.Methods①The cDNA fragment of HCV core region was generated by the reverse-transcriptase-polymerase chain reaction (RT-PCR), and the plasmid pGEM-HCV was constructed to generate in vitro transcripts of core region of HCV RNA genome. Dig-labeled HCV C-RNA transcripts and unlabeled HCV C-RNA transcripts were obtained by in vitro transcription. After binding of HCV C-RNA and protein extracts from HepG2 cells, electrophoretic mobility shift assay (EMSA), Ultraviolet (UV) cross-linking experiment and competition analysis were performed to screen HepG2 cellular proteins, which interact with DIG-labeled transcripts of core region of HCV RNA genome. After electrophoresed on PAGE, proteins or RNA were transferred to NC membrane. DIG-labeled complexes were detected with anti-Digoxingenin-AP in order to find whether cellular proteins bound to HCV C-RNA or not.②The different HCV C-RNA transcripts in length were preparated by PCR, cloning and in vitro transcription. After binding of the different HCV C-RNA transcripts and protein extracts from HepG2 cells, UV cross-linking experiment was performed to identify binding region of HCV C-RNA. DIG labeled RNA-binding proteins were separated by immunoprecipitation with anti-DIG. By comparing SDS-PAGE’s results with NC membrane’s resluts, the proteins bands were excised from SDS-PAGE and were analyzed by MALDI-TOF-MS.③Total RNA was isolated from HepG2 cells, and the entire coding region of PGAB-B cDNA was obtained by RT-PCR. The cDNA fragment of PGAM-B was cloned into the pcDNA?3.1/V5-HisA, and the eukaryotic expression plasmid pcDNA-PGAM was constructed. The cDNA fragment of HCV core protein was obtained by PCR from plasmid pGEM-HCV and was cloned into pcDNA3.0 to construct the eukaryotic expression plasmid pcDNA-HCV. After identified by restriction endonuclease and sequencing,the eukaryotic expression plasmids were transfected into HepG2 cells respectively. The instantaneous expression of PGAM-His and HCV core protein were analyzed by immunocytochemical technique. Results①503 bp cDNA fragment of HCV core gene was obtained from HCV carrier. The plasmid pGEM-HCV was constructed and identified by PCR, restriction endonuclease and sequencing.②HCV cDNA sequence of core gene was compared with the known HCV gene sequence (AB092962.1) in GenBank. There were only 2 nucleotide differences between the two sequences, which located in position nt 67 (23aa K→E) and nt 177 (silent mutation).③By primary screening of EMSA, there were cellular proteins of HepG2 cells bingding to HCV C-RNA.④By screening of UV cross-linking experiment, there were several cellular proteins of HepG2 cells bingding to HCV C-RNA in vitro. The intensity of RNA binding protein bands increased with increasing amounts of cell extracts. The binding of cellular proteins to Digoxin labeled HCV C-RNA was competed out in proportion to the increasing amount of unlabeled HCV C-RNA.⑤Three different HCV C-RNA transcripts (198nt, 306nt and 503nt) could bind to cellular proteins in vitro, but the binding band of HCV C198 RNA to cellular proteins was clearer and sharper than that of others.⑥After immunoprecipitated, four RNA binding proteins were detedted in NC membrane. The protein band of P30 was the clearest, and the protein amount was also the largest.⑦The P30 protein bands were excised from SDS-PAGE and analysed by MALDI-TOF-MS. The P30 protein was identified as PGAM-B (Phosphoglycerate mutase isozyme B).⑧760bp fragment of PGAM-B cDNA sequence was obtained by RT-PCR from HepG2 cells, and it was consistent with the known PGAM-B sequence (J04173) in GenBank.⑨After the eukaryotic expression plasmids pcDNA-PGAM and pcDNA-HCV were transfected into HepG2 cells, the fusion protein PGAM-His and HCV core protein were instantaneous expressed in some of cells. Conclusions①There are several cellular proteins could specifically bind to core region of HCV RNA genome in vitro.②The binding site of HCV C-RNA maybe located in 5’-terminal of core region of HCV RNA genome.③PGAM-B could specifically bind to the core region of HCV RNA genome in vitro. It suggested that PGAM-B maybe involved in translation and replication of HCV RNA by interacting with core region of HCV RNA genome.④PGAM-B and HCV core peotein couled instantaneous express in HepG2 cells.更多还原