【作者】 朱峰；

【导师】 周天鸿；

【作者基本信息】 暨南大学 ， 细胞与基因工程， 2009， 博士

【摘要】 背景与目的人巨细胞病毒UL49开放阅读框(ORF)为疱疹病毒保守基因。目前,除Walter Dunn等利用细菌人工重组染色质(BAC)技术发现UL49缺失突变病毒不能再包装,间接证实UL49为人巨细胞病毒生长的必须基因外,对UL49相应功能研究均局限于生物信息学分析与推测。为了进一步证实UL49在病毒生物学方面的功能,有必要首先阐明以下问题：UL49的mRNA表达及全长cDNA精确序列；能否检测到UL49编码的蛋白(pUL49)、确定pUL49表达时相及蛋白特性(病毒结构蛋白、还是非病毒结构蛋白?)等；在此基础上,对以下问题进行实验论证：pUL49在病毒感染宿主细胞中的定位以及在病毒感染、复制中的生物学功能；pUL49可能与宿主细胞哪些蛋白分子相互作用等。方法将HCMVA接种生长良好的人胚肺成纤维细胞(HELF),抽提病变细胞中的病毒RNA;以SMART RACE(Rapid Amplification of cDNA Ends, RACE)技术扩增UL49的5’和3’末端,TA克隆两类RACE产物,测序；用RT—PCR扩增UL49全长cDNA序列。获取Genebank公布的UL49编码氨基酸序列,用在线软件对其Hopp&Woods亲水性、可及性、极性及柔韧性、Welling抗原性和二级结构等参数法进行分析,用吴玉章等建立的B细胞表位预测法综合评价；以化学方法对预测片断进行多肽合成、纯化、与载体血兰蛋白(KLH)联接；将多肽-血兰蛋白化合物(免疫原)免疫新西兰大白兔,获得兔免疫血清后,用ELISA测定效价；构建HCMV UL49大片断(aa3-246)原核表达载体pGEX-4T-3-UL49A,诱导表达pUL49A-GST融合蛋白；构建HCMV UL49全基因真核表达载体pCDNA3.1-UL49-myc,瞬时表达pUL49-myc融合蛋白；以包装成功的HCMV-Towne病毒株感染HFF细胞,收集7天后的培养上清液,以anti-HCMV pUL49为一抗进行上述三种样本Western blotting实验。以RV-Towne感染HFF,收集3、6、12、24、48、72、96、120hr后细胞裂解液,以anti-HCMV pUL49、anti-IE (CMVpp72/86)、anti-E (CMVgB/CH28)、anti-LA (CMVpp28/CH19)分别对细胞裂解上清液进行WB；收集接近100%细胞病变的HFF[RV-Towne]培养上清液(病毒颗粒),于58750×g、4℃离心纯化病毒,分别用Triton X-100、Trypsin预处理并4℃、100000g离心,以可溶混合上清液、不溶蛋白部分分别进行Western blotting。用试剂盒抽提、纯化重组人巨细胞病毒Towne基因组(Towne-BAC)、delUL49 Towne基因组(delUL49 Towne-BAC),构建pcDNA3.1(+)-UL82表达质粒；将Towne-BAC、delUL49 Towne分别与pcDNA3.1 (+)-UL82共转HFF,将Towne-BAC、delUL49 Towne分别与pcDNA3.1 (+)-UL82、pcDNA3.1 (+)-UL49共转HFF,培养病毒、观察细胞病变；以RV-Towne感染HFF,收集3、6、12、24、48、72、96、120hr后细胞,分别用Hochest33258、anti-HCMVpUL49、anti-E、anti-LA进行细胞免疫影像实验,荧光显微镜下观察、影像合成定位分析；将纯化的重组病毒与anti-pUL49抗体经37℃预处理0.5hr后,以1 PFU／cell量感染HFF细胞,收集1-7天的病毒细胞上清夜,再观察其生长状况；将上述预处理RV-Towne以1 PFU／cell量感染HFF细胞,收集1、3、6hr的病毒感染细胞,抽提总染色质DNA,以连续稀释后的总染色质DNA为模板,PCR扩增RV-Towne特异基因IE1序列,半定量分析RV侵入细胞的量。扩增UL49全序列,克隆入pGBKT7,将pGBKT7-UL49转化到酵母细胞AH109,用蛋白印迹法分析诱饵蛋白的表达,检测诱饵蛋白有无毒性和自激活效应；将人胚肾细胞cDNA文库与pGBKT7-UL49共转AH109,于营养缺陷型培养基中筛选阳性克隆,按文献将阳性克隆酵母作β-半乳糖苷酶活性测定；将酵母质粒电转DH5α,抽提质粒,设计文库插入片段上游引物,对双杂交阳性且β-半乳糖苷酶阳性细菌质粒的AD插入片段进行PCR；将非重复克隆细菌质粒的AD插入片段PCR扩增产物进行HaeⅢ酶切,将非重复克隆质粒进行测序,通过Genebank同源性搜索和序列比对分析；选取同源性搜索和序列比对分析后的基因重复酵母双杂交及其β-半乳糖苷酶活性实验；扩增CYB5D2、C11ORF17、COL3A1、UCHL3因片断,构建其pGEX4T-3的表达质粒,构建pCDNA3.1(+)-UL49真核表达质粒；用IPTG诱导细菌表达CYB5D2、C11ORF17、COL3A1、UCHL3的GST融合蛋白,瞬时表达pUL49蛋白,按文献将pUL49分别与CYB5D2、C11ORF17、COL3A1、UCHL3的GST融合蛋白进行GST-pull down；构建pCDNA3.1(+)-COL3A1、pCDNA3.1(+)-C11ORF17真核表达质粒,分别瞬时转染HFF细胞,以1.0PFU/cell量感染上述两组细胞,收集感染1-7后的细胞培养上清夜进行病毒生长实验。结果HCMVA在体外成功感染HELF,感染的HELF中有HCMV的IE1和LA标志性基因；用SMART RACE技术从病毒RNA中获得UL49基因的5’和3’UTR,分别克隆后测序,UL49基因5’UTR长90bp、3’UTR长315bp；RT-PCR扩增获得2118bp的UL49基因全长cDNA序列,其开放读码框编码570个氨基酸。多种预测法重复了人巨细胞病毒pUL49蛋白的N端第228～243位氨基酸区域内或附近,该区域含有β转角和无规卷曲结构；化学方法合成得到了该区域氨基酸序列KRFDARADLAVY-KLH的免疫原；获得免疫兔血清,ELISA测定效价达1：8000以上；以此anti-HCMV pUL49为一抗得WB实验显示pUL49A-GST融合蛋白、pUL49A-myc融合蛋白、HFF[RV-Towne]细胞蛋白裂解液均显示蛋白条带,其分子量与理论值吻合。RV-Towne感染HFF在12hr后,pUL49开始出现表达,至RV-Towne感染96hr达峰值,与典型的HCMV时相基因pp72／86、gB、pp28的表达对照,类似gB蛋白时相；用细胞表面活性剂TX-100以及蛋白胰酶对纯化病毒进行不同处理后,pUL49与pp28的WB实验结果一致,而与gB存在明显差异。成功构建pcDNA3.1(+)一UL82.pcDNA3.1(+)-UL49；Towne-BAC与pcDNA3.1(+)-UL82共转15天后可见绿色荧光,delUL49 Towne-BAC与pcDNA3.1(+)-UL82共转30天后仍未见绿色荧光；Towne-BAC与pcDNA3.1(+)-UL82、pcDNA3.1(+)-UL49共转15天后也可见绿色荧光,delUL49 Towne-BAC与pcDNA3.1(+)-UL82共转20天后方见绿色荧光,至30天后荧光强度仍较Towne-BAC的结果相距甚远；病毒感染HFF细胞过程中,pUL49出现时间与蛋白表达出现时间一致,均出现于12hr,影像显示pUL49定位于细胞质而非细胞核,且在细胞感染24hr内显现均匀散布状,而后渐成聚集状,定位于核外边界某一点状区域、且量逐渐增多；以早期表达蛋白gB、晚期表达蛋白pp28为对照,pUL49与pp28在72、96hr存在共定位,而与gB虽然在24、72、96hr均出现表达影像,但未发现两者之间的共定位；经anti-pUL49预处理的重组病毒在感染前2天与未经处理的病毒出现近1个数量级的生长差异,两者在其后的差异均在1个数量级内,差异并不明显；anti-pUL49预处理的病毒在感染侵入阶段(1-6hr),RV-Towne特异基因IE1序列PCR扩增产物与未经anti-pUL49预处理的病毒差异不显著。克隆成功pGBKT7-UL49并成功转化到AH109,AH109[pGBKT7-UL49]表达诱饵蛋白pUL49,pUL49对转化细胞无细胞毒性、无自激活；酵母双杂交筛选得到30个SD／-Trp-Leu-His-Ade阳性且β-半乳糖苷酶阳性克隆,PCR、HaeⅢ酶切鉴定结果显示11个非重复克隆质粒；其中4个基因重复酵母双杂交及其β-半乳糖苷酶活性,显示C11ORF17、COL3A1表达蛋白与pUL49间的相互作用相对较强；诱导细菌表达pGEX4T-3-CYB5D2、pGEX4T-3-C110RF17、pGEX4T-3-COL3A1、pGEX4T-3-UCHL3的GST融合蛋白与pUL49瞬时表达蛋白相互对应的GST-pull down,结果显示C11ORF17、COL3A1出现条带呈现阳性结果,而CYB5D2、UCHL3未出现条带；pC11ORF17表达的宿主细胞实验组重组病毒生长受到一定抑制,而pCOL3A1表达的宿主细胞实验组重组病毒生长与未处理组无显著差异。结论在体外HCMVA感染过程中,UL49的mRNA存在表达,其cDNA序列全长共2118bp,其中5’UTR长90bp,3’UTR长315bp,UL49开放阅读框编码570个氨基酸。多种预测法重复了pUL49蛋白的N端第228-243位氨基酸区域内或附近为B细胞识别区；用化学合成得到KRFDARADLAVY-KLH免疫原,获得效价高、特异性强的兔anti-HCMV pUL49抗血清。人巨细胞病毒UL49开放阅读框编码蛋白在病毒感染过程中存在表达,且pUL49为早期表达蛋白；pUL49与pp28对TX-100、蛋白胰酶的耐受相近,暗示两者定位于病毒颗粒同一区域(皮层)pUL49为Towne的生长必需基因；在表达pUL49的HFF细胞中,缺失UL49的Towne能部分得到回复生长；病毒感染过程pUL49定位于细胞质而非细胞核,随感染时间的持续有被招募于宿主细胞特定区域的趋势；pUL49与pp28在72、96hr存在共定位,暗示pUL49在感染晚期定位于细胞内质网；在病毒增殖过程中,anti-pUL49影响了病毒生长速度,但在感染过程中,未影响病毒的侵入环节。成功构建了酵母诱饵表达载体pGBKT7-UL49,经双杂交系统筛选,成功获得11个非重复克隆质粒；其中C110RF17、COL3A1表达蛋白与pUL49间的相互作用相对较强；GST-Pull down验证了该两个蛋白与pUL49的体外相互作用；pUL49与pC110RF17的相互作用可能影响病毒的增殖与生长,而pUL49与pCOL3A1的相互作用对病毒的增殖与生长基本无影响。更多还原

【Abstract】 Background and objectivesUL49 is a herpes virus conserved Open Reading Frame (ORF).Up to now, besides that Walter Dunn and his team found UL49 was a essential gene for HCMV growth when they recovered the bacterial artificial chomosome containing human cytomegalovirus genome deleted UL49 ORF, the study of UL49 gene function limited only to the biophysical information analysis. For studying the biophysical function of UL49 gene during HCMV infection, it is necessary to unclose firstly the question as below:weather is the mRNA expression and how are the cDNA sequences? Can detect the UL49 ORF encoding protein? When do the protein express and where do the protein locate in virion or host cells? After answering the above question, we will observe pUL49 location in host cells infected HCMV and the biophysical function of pUL49 during HCMV infection and production, and also observed the target proteins interacting with the bait protein pUL49.MethodsHCMV AD 169 (HCMVA) was inoculated to well growth primary human embryonic lung fibroblasts, the HELFs were collected while the cytopathic effect appearing, and HCMVA RNA was extracted from the collected cells.5’RACE and 3’RACE of cDNA of HCMV UL49 were amplified by SMART rapid amplification of cDNA ends technology. The full lengh cDNA of HCMV UL49 was amplified by RT-PCR, and cloned the cDNA into pMD18-T simple vector and sequenced subsequently. The secondary structure and transmembrane domain were predicted by SOPMA and TMHMM respectively. Hydrophilicity, accessibility, polarity, flexibility, surface probability and antigenicity index predicted by methods of Kyte&Doolittle, Emini, Zimmerman and average flexibility. EMBOSS and Bopped Wu’method were combined and the possible B cell epitopes of pUL49 protein were predicted. The B cell epitome of pUL49 protein was synthesized and purified using chemical method then linked it with KLH and condensed with EDC to form immunogen KRFDARADLAVY-KLH. When New Zealand Rabbits were immuned by the immunogen. After collected serum from the immuned rabbits, the serum titer was detected using ELISA. pGEX-4T-3-UL49A from HCMV UL49 (aa3-246) was contracted and pUL49A-GST was induced using IPTG, pCDNA3.1-UL49A-myc from HCMV UL49 was contracted and the pUL49A-myc protein was obtained using instantaneous transfection method. Cell-free supernatants were collected from the medium infected HCMV seven days later. The pUL49 was finally detected from above three samples using Western blotting.Human foreskin fibroblasts (HFFs), after infected RV-Towne 3,6,12,24,48,72,96 and 120 hours, were collected and cracking, samples were confirmed by Western blotting using the monoclonal antibody anti-IE (CMVpp72/86,CH160:sc-69748, Santa Cruz Biotechnology, Inc.), anti-E (CMV gB, CH28, sc-69742, Santa Cruz Biotechnology, Inc.), anti-LA (CMVpp28, CH19, sc-69749, Santa Cruz Biotechnology, Inc.) and anti-HCMV pUL49. HCMV virion were collected from the medium HFFs appearing near 100% cytopathic effect, and purified using 58,750xg at 4℃.The purified virion were pretreated with triton X-100 or trypsin, then treated with 100000 g at 4℃. The pUL49 from above samples were finally detected using WB.HCMV BACs were extracted and purified by using a Nucleobond AX kit. The fragment of UL82 was amplified by PCR and cloned into the vector pcDNA3.1 (+). The vector pcDNA3.1 (+)-UL82 or pcDNA3.1 (+)-UL82 and pcDNA3.1 (+)-UL49 together with BACs were co transformed into HFFs respectively. Confluent cytopathic effect was observed from the infected cells by microscope. HFFs, after infected RV-Towne 3,6,12,24,48,72,96 and 120 hours, were collected and confirmed by immunohistochemistry using the monoclonal antibody anti-E, anti-LA, hochest33258 and anti-HCMV pUL49. The cells images were observed by fluorescence microscope. The purified RV were treated with anti-pUL49 at 37℃for 30 minutes, then infected HFFs with 1 PFU/cell. The cell-free supernatants after infected 1-7 days were collected to observe the growth curve. As above, the cells after infected 1-6 hours were collected and extracted total DNA from these cells, then amplified RV-Towne special gene IE1 and analyzed the product titer. The fragments of UL49 was amplified by PCR, and then cloned into the bait expression vector pGBKT7. The bait vector pGBKT7-UL49, being verified by sequencing, was transformed into AH 109 yeast cells. Then Western Blotting analyzed the bait protein pUL49. Toxicity and self-activation of the bait protein were detected by cultured in different SD cultures. Human embryonic kidney cells CDNA library together with pGBKT7-UL49 were co transformed into yeast cells AH109. The positive clones were selected in different medium deleted nutrient, and detected by filter assay. The positive vectors were transformed to DH5 a and extracted plasmid.Designed PCR primer of cDNA library vector, and the positive gene sequences were amplified, the PCR products also digested by HaeⅢ. The positive sequences were identified by DNA sequence analyzer. With Genebank BLAST and other analysis software, the positive genes were recovered by hybrid and filter assay. Amplified the genes CYB5D2, C11ORF17, COL3A1 and UCHL3, the four fragments were cloned into the vectors pGEX4T-3, pCDNA3.1 (+)-UL49 was constructed meantime. The fusion proteins of CYB5D2, C11ORF17, COL3A1 and UCHL3 with GST were induced by IPTG respectively, pUL49 was expressed also. According to reference, pUL49-3Xflag together with the GST fusion CYB5D2, C11ORF17, COL3A1 and UCHL3 proteins were done GST-pull down test respectively. pCDNA3.1(+)-COL3A1 and pCDNA3.1(+)-C11ORF17 were constructed, and they were transformed into HFFs respectively,24 hours later the cells were infected RV with 1.OPFU/cell. The cell-free supernatants after infected 1-7 days were collected to observe the RV growth curve. The cell-free supernatants after infected 1-7 days were collected to observe the RV growth curve.ResultsWe infected HCMVA successfully to well growth HELFs. and HCMV typical genes IE1 and LA were identified using PCR. Using Smart assay, we obtained and cloned the 3’and 5’termination of UL49 gene. Sequencing results suggested its 3’- untranslated region and 5’-untranslated regions were 315bp and 90bp respectively. The full-length cDNA of UL49 gene,2118bp, was amplified using RT-PCR.The pUL49 protein N-terminal amino acid residues locating at 228-243 regions were predicted by all prediction methods, and this regions containedβsheet and coils structure, which regions or their surrounding regions were the predominant epitomes. The B cell epitomes of pUL49 protein were synthesized and purified, linked with KLH and condensed successfully with EDC. Immunogen KRFDARADLAVY-KLH was formed finally. After immuned, immuned rabbit serum was collected and then detected the titer being 1:8000. pGEX-4T-3-UL49A (3-246) was contracted and the pUL49A-GST was induced, pCDNA3.1-UL49 was contracted and pUL49-myc protein was obtained, the pUL49 from above three samples were detected.pUL49 protein began to express after HFF infected RV-Towne 12 hours and the maxim titer appeared after infected RV-Towne 96 hours. pUL49 expression kinetics was related with the type HCMV gene gB.pUL49 WB image was related with the type HCMV gene pp28 after the samples treated with TX-100 and trypsin.We succeed in constructed the pcDNA3.1(+)-UL82 and pcDNA3.1(+)-UL49, the HFFs co transformed with pcDNA3.1(+)-UL82 or together with pcDNA3.1(+)-UL49 and Towne-BACs were observed green fluorescence 15 days later. pcDNA3.1(+)-UL82 together only with pcDNA3.1(+)-UL49 and delTowne-BACs were observed green fluorescence 20 days later. pUL49 appeared red fluorescence in infected cells 12 hours later and located in cytoplasm, and pUL49 appeared from spreading uniformly before infected 24 hours to condense somewhere region. pUL49 was also collocated with pp28 at infected 72-96 hours. The growth curve of RV treated with anti-HCMV pUL49 reduced 10 times than untreated with anti-HCMV pUL49. The DNA of RV treated with anti-HCMV pUL49 was not different to untreated with anti-HCMV pUL49 during virus invasive period.UL49 was amplified and cloned into pGBKT7 successfully. The vector pGBKT7-UL49 was transformed into AH 109 as well and those cells exhibited neither toxicity nor self-activation. Western Blotting detected the expression of the bait protein pUL49. Human embryonic kidney cells CDNA library together with pGBKT7-UL49 were co transformed into yeast cells AH 109.30 positive clones were selected in different medium deleted nutrient and filter assay.11 none repeated positive vectors were identified by PCR and HaeⅢdigestion.4 positive genes were recovered hybrid and filter assay, gene C11ORF17 and COL3A1 appeared stronger ability to interact with the bait protein pUL49. The vectors pGEX4T-3 with above four fragments were constructed, pCDNA3.1 (+)-UL49 was constructed meantime. The fusion GST proteins, pUL49 protein were expressed. pC11ORF17 and pCOL3A1 proteins appeared positive pull down test result. pCDNA3.1(+)-COL3A1 and pCDNA3.1(+)-C11ORF17 were constructed. The HFFs expressed pCDNA3.1(+)-C11ORF17 appeared lower virus growth than normal HFFs, but the pCDNA3.1(+)-COL3A1 appeared no affection virus growth to normal HFFs.ConclusionWe observed the mRNA expression of HCMV UL49. The cDNA sequence were 2118bp in size, and its 5’-untranslated region were 90bp, while its 3’-untranslated region were 315bp. HCMV UL49 open reading frame were 1710bp and capable of encoding 570 amino acids.Prediction of the secondary structure and B cell epitopes of pUL49 protein laid the foundation for studying the characteristics of the protein and developing the epitope-based the monoclonal antibody against pUL49 protein. ImmunogenKRFDARADLAVY-KLH was synthesized to the B cell epitopes of pUL49 protein. We obtained high titer and strong specific rabbit serum anti-HCMV pUL49. pUL49, HCMV UL49 ORF encoding protein, expressed in the host cells infected HCMV, and its expression kinetic was early period. That pUL49 endured TX-100 and trypsin was near to pp28, these two proteins maybe located one region in virion. We succeed in recovering the recombinant HCMV (RV) from the recovered BACs containing HCMV genome. pUL49 was the essential gene for Towne growth. In HFFs expressed pUL49. delTowne-BACs only recovered partly. pUL49 was located at cytoplasm in infected cells and was recruited to somewhere region during infected later period. pUL49 appeared collocated in endoplasmic reticulum with pp28 at 72-96 hours.Anti-pUL49 affected the growth of RV, but did not affect virion entry to its host cells.The bait expression vector of UL49 was constructed successfully, which laid the foundation for screening target proteins interacting with the bait protein pUL49 using the yeast two-hybrid technique. With hybrid test, we obtained 11 none repeated positive vectors. From 4 positive genes, gene C11ORF17 and COL3A1 appeared stronger ability to interact with the bait protein pUL49. Which result was reinsured by GST pull down test. The interaction between pUL49 with pC11ORF17 maybe affects growth and proliferation of HCMV in HFFs, but the interaction between pUL49 with pCOL3A1 maybe not affects growth and proliferation of HCMV in HFFs.更多还原