鸭瘟病毒UL49基因功能研究
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摘要
1、鸭瘟病毒UL49基因生物信息学分析。本章根据实验室测定的DPV UL49基因核酸序列及氨基酸序列,对其进行了生物信息学分析,研究结果表明：DPVUL49基因编码VP22蛋白,此蛋白由253aa组成,分子量约为27864.575Da,其中Arg、Ala、Ser、Asp与Thr的含量最高,分别为11.85%、11.85%、11.06%、7.11%及7.11%。相似性及进化树分析表明DPV应该划分为α疱疹病毒亚科、马立克病毒属。DPV VP22蛋白无信号肽、无跨膜区、无核输入及核输出信号,等电点为9.795,其C端126aa～191aa区域保守性最强；VP22蛋白可能含有2个N-糖基化位点、3个cAMP和cGMP-依赖蛋白激酶磷酸化位点,9个蛋白激酶C磷酸化位点,3个酪氨酸激酶Ⅱ磷酸化位点及3个酰胺化功能位点。亚细胞定位分析表明,DPV VP22蛋白存在于细胞核、细胞浆及线粒体中的可能性分别为87.2%、12.7%和0.1%。
2、鸭瘟病毒UL49基因克隆、原核表达、蛋白纯化及多克隆抗体制备。根据实验室提供的DPV UL49基因序列,采用Oligo6.71软件设计了一对特异性引物,并将扩增出的UL49全基因克隆入pMD18-T载体中,经BamH I和Hind III双酶切后,克隆入原核表达载体pET-28a(+)中,成功构建了pET28a-UL49重组原核质粒。将此质粒转化表达宿主菌BL21(DE3),经过对IPTG浓度、诱导温度及诱导时间的优化,确定了pET28a-UL49重组质粒表达的最佳条件：即在37℃条件下,0.6mmol/L IPTG诱导表达4h,表达的重组VP22蛋白约为35kDa,具有高度可溶性及很好的反应原性。将纯化后的重组VP22蛋白免疫雄性家兔,制备分离兔抗重组VP22蛋白高免血清,效价最高为1：32；经饱和硫酸铵法粗提、HighQ阴离子交换层析法纯化后,、Western blot检测能够与纯化的VP22重组蛋白结合,具有很强的特异性,为进一步研究VP22蛋白的功能奠定了基础。
3、鸭瘟病毒VP22蛋白亚细胞定位研究。采用间接免疫荧光法研究了DPV VP22蛋白在病毒感染鸭胚成纤维细胞中的定位时相,结果表明：DPV感染细胞6h后,在胞浆、核膜周围可见特异性荧光；12h后,在胞浆、核内均可见特异性荧光,胞核中的荧光呈均匀分布；随着时间推移,在病毒感染24h和36h后,绝大多数细胞核内都出现荧光且逐渐增强,胞浆中的荧光渐趋减少。由于在感染晚期,DPV可使鸭胚成纤维细胞形成蚀斑,细胞死亡后发生脱落进入培养液中,故在48h和72h时表达荧光的细胞数量渐趋减少。此外,大肠杆菌表达的VP22重组蛋白能够由培养液自主进入细胞内部：VP22蛋白加入MEM培养液4h后,胞浆内部可发现特异性荧光,并且随着时间的推移,胞浆内的荧光越来越强,至36h时几乎所有细胞的胞浆内均可见特异性荧光并呈颗粒状分布,荧光强度达到最大；可是在胞核内始终未见特异性荧光。
4、鸭瘟病毒UL49基因转录时相与表达时相分析。本试验采用荧光定量PCR法及Western blot法研究了DPV UL49基因在鸭胚成纤维细胞中的转录及表达时相,试验结果表明：DPV UL49基因在病毒感染1h后开始转录,12h后转录量开始明显增加,24h时采用Western blot法能够检测到VP22蛋白,至72h～84h时UL49基因的转录量和表达量均达到最大值,之后开始逐渐降低。
5、鸭瘟病毒VP22蛋白核定位信号研究。为了研究DPV VP22蛋白是否存在核定位信号及其功能位点的位置,以绿色荧光蛋白为标记,构建了一系列不同区域截短VP22蛋白缺失突变体与GFP融合表达的真核质粒,然后使用脂质体介导转染鸭胚成纤维细胞,通过荧光显微镜观察分析各个缺失突变体的亚细胞定位,从而确定与核定位相关的结构域,为进一步深入研究DPV VP22蛋白的结构与功能奠定了基础。试验结果表明：(1)DPV VP22蛋白具有核定位功能,并且这种功能不依赖其他DPV蛋白；(2)DPV VP22蛋白C端165aa～253aa区域能够独立作为核定位信号；(3)DPV VP22蛋白的核定位功能在活细胞及不同固定剂处理后的细胞中都能够检测到,所以其核定位功能不受固定剂的影响。
6、DPV UL49基因RNA干扰有效靶点筛选及其对病毒重要糖蛋白基因转录的影响。根据实验室提供的DPV UL49基因序列,由上海吉玛制药技术有限公司设计4个不同的shRNA序列,然后插入到pGPU6载体中。将4个RNAi真核质粒与第六章构建的VP22a-GFP荧光质粒共转染鸭胚成纤维细胞,荧光显微镜观察干扰结果,4个RNAi干扰质粒均能够抑制VP22a-GFP质粒表达荧光,其中VP22-505和VP22-555表现的抑制作用最强。4种RNAi质粒对DPV UL49基因转录具有不同程度的抑制作用,尤其以VP22-505抑制效率最高,达到80%,从而确定了UL49基因中的“5-GCTGTTGGAGATTGCCAATGA-3"序列(即505bp-525bp)是RNAi发挥作用的最佳靶点。RNAi质粒VP22-505对DPV UL49基因转录抑制的同时,对DPV糖蛋白gB、gD和gE基因的转录也具有很强的抑制作用。
1. Bioinformatic analysis of UL27gene of Duck Plague Virus. In this paper, we conducted a detailed bioinformatic analysis of VP22protein. Our results showing that the DPV VP22protein has a molecular weight of27864.575Da, its pHi is computed to be9.795. The contents of Arg, Ala Ser, Asp and Thr were11.85%,11.85%,11.06%,7.11%and7.11%, respectively. For DPV VP22protein, the carboxyl terminus (126aa～191aa) is highly conserved. Similarity comparison and phylogenetic analysis showed that DPV aligns into the Alphaherpesvirus subfamily and is closely related to MDV. Two N-glycosylation sites, three cAMP-and cGMP-dependent protein kinase phosphorylation sites, nine protein kinase C phosphorylation sites, three Casein kinase II phosphorylation sites, two N-myristoylation sites, three Amidation sites were determined by PROSCAN. YLoc predicted that the DPV VP22protein sequence is located in the nucleus, cytoplasm and mitochondria with a probability of87.2%,12.7%and0.1%, respectively. Our results may be useful in future studies to elucidate the biological functions of VP22during DPV infection.
2. Cloning, prokaryotic expression, purification and polyclonal antibody preparation of DPV UL49gene. The full-length UL49gene of DPV was amplified with a pair of primer. This product was cloned into the multiple cloning site of the pMD19-T vector. For recombinant protein production, a prokaryotic expression vector pET28a(+) was used. pMD19-UL49was digested with BamH Ⅰ and Hind Ⅲ and the UL49sequense was inserted into the BamH Ⅰ/Hind Ⅲ site of pET-28a(+), which is capable of producing recombinant protein with an N-terminal6xHis tag. The result was designated pET28a-UL49and transformed into E. coli BL21(DE3) for recombinant protein production by calcium chloride method. For optimizing protein expression, cells were harvested4h after induction by0.6mM IPTG at37℃may prove optimal for the yield of soluble protein. For the production of the polyclonal rabbit anti-VP22serum, a adult New Zealand white rabbit was inoculated four times with VP22protein. The rabbit was bled7to10days after the final booster, then it was killed and the IgG fraction of the serum was isolated by ammonium sulfate precipitation. Then, using a DEAE-Sepharose column, the IgG fraction was purified by ion-exchange column chromatography. These results will provide a basis for further analysis of the DPV VP22protein. Western blots shows that the rabbit anti-VP22polyclonal antibody has good specificity.
3. Subcellular localization of Duck Plague Virus protein VP22. Indirect immunofluorescence tests revealed that the VP22antigens is detectable as early as6h postinfection, it exists predominantly in the cytoplasm and perinuclear region of DPV infected DEF in a diffuse fluorescent pattern early in infection, then migrates to and accumulates in the nucleus at12hpi, virtually every cell in the culture exhibited bright nucleus fluorescence at36hpi. At late stages of infection, cells expressing fluorescence dramatically reduced due to plaque famation at48hpi and72hpi. VP22found in infected cells is distributed in at least three distinct subcellular localizations, which we define as cytoplasmic, diffuse, and nuclear. Results of immunofluorescence studies show that the VP22recombinant protein can be detected in DEF after4h incubation in MEM, exhibiting pronounced accumulation in the cytoplasm, but failed migrates to and accumulates in the nucleus, virtually every cell in the culture exhibited bright cytoplasm fluorescence at36h. These results indicate that purified VP22recombinant protein retains the import property.
4. Transcription characteristics and expression kinetics analysis of Duck Plague Virus UL49gene. In this study, we determined the transcription characteristics and expression kinetics of DPV UL49gene by FQ-PCR and Western blot method. The results showed that DPV UL49gene transcripts appeared at1hpi, the relative expression level was at a low level in the first12hpi, and VP22protein can be detected at24hpi, and then the transcripts and VP22protein were peaked at72-84hpi, thereafter both of them declined.
5. Identification of the nuclear localization signal of Duck Plague Virus VP22protein. In order to mapped the functions of DPV VP22to specific regions in the polypeptide, we constructed a series of deletion constructs of VP22tagged by the GFP. The DPV VP22derivatives was transfected into DEF cells as described in Materials and Methods, and the subcellular localization pattern of the fusion proteins were examined using a Nikon Eclipse TE2000-U Inverted Microscope or a Nikon ECLIPSE80i fluorescence microscope. In this report, we demonstrate for the first time that (1) the nuclear localization of VP22is independent of other viral proteins,(2) VP22does not have a classical NLS, but a89-amino acid sequence from165aa to253aa within the carboxyl terminus was sufficient for nuclear localization.(3) the nuclear localization of VP22is independent of fixing agent.
6. The determination of RNA interference target for DPV UL49gene and its transcriptive effect to glycoprotein genes. According to the DPV UL49gene sequence,4shRNA were designed and constructed in pGPU6plasmids by Shanghai GenePharma Co.,Ltd.4RNAi plasmids and VP22a-GFP were cotransfected in DEF cells using LipofectamineTM2000Reagent, the results showed that VP22-505and VP22-555have inhibitive effect. Relative quantitative real-time RT-PCR analysis of UL49mRNA levels throughout DPV infections showed the "5-GCTGTTGGAGATTGCCAATGA-3" sequence (505bp～525bp) can be seen as the best target spot. When DEF cells were transfected with VP22-505expression vectors and then infected with DPV, the transcriptional effect of UL49, gB, gD and gE can be severely down-regulated by VP22-505.
2、鸭瘟病毒UL49基因克隆、原核表达、蛋白纯化及多克隆抗体制备。根据实验室提供的DPV UL49基因序列,采用Oligo6.71软件设计了一对特异性引物,并将扩增出的UL49全基因克隆入pMD18-T载体中,经BamH I和Hind III双酶切后,克隆入原核表达载体pET-28a(+)中,成功构建了pET28a-UL49重组原核质粒。将此质粒转化表达宿主菌BL21(DE3),经过对IPTG浓度、诱导温度及诱导时间的优化,确定了pET28a-UL49重组质粒表达的最佳条件：即在37℃条件下,0.6mmol/L IPTG诱导表达4h,表达的重组VP22蛋白约为35kDa,具有高度可溶性及很好的反应原性。将纯化后的重组VP22蛋白免疫雄性家兔,制备分离兔抗重组VP22蛋白高免血清,效价最高为1：32；经饱和硫酸铵法粗提、HighQ阴离子交换层析法纯化后,、Western blot检测能够与纯化的VP22重组蛋白结合,具有很强的特异性,为进一步研究VP22蛋白的功能奠定了基础。
3、鸭瘟病毒VP22蛋白亚细胞定位研究。采用间接免疫荧光法研究了DPV VP22蛋白在病毒感染鸭胚成纤维细胞中的定位时相,结果表明：DPV感染细胞6h后,在胞浆、核膜周围可见特异性荧光；12h后,在胞浆、核内均可见特异性荧光,胞核中的荧光呈均匀分布；随着时间推移,在病毒感染24h和36h后,绝大多数细胞核内都出现荧光且逐渐增强,胞浆中的荧光渐趋减少。由于在感染晚期,DPV可使鸭胚成纤维细胞形成蚀斑,细胞死亡后发生脱落进入培养液中,故在48h和72h时表达荧光的细胞数量渐趋减少。此外,大肠杆菌表达的VP22重组蛋白能够由培养液自主进入细胞内部：VP22蛋白加入MEM培养液4h后,胞浆内部可发现特异性荧光,并且随着时间的推移,胞浆内的荧光越来越强,至36h时几乎所有细胞的胞浆内均可见特异性荧光并呈颗粒状分布,荧光强度达到最大；可是在胞核内始终未见特异性荧光。
4、鸭瘟病毒UL49基因转录时相与表达时相分析。本试验采用荧光定量PCR法及Western blot法研究了DPV UL49基因在鸭胚成纤维细胞中的转录及表达时相,试验结果表明：DPV UL49基因在病毒感染1h后开始转录,12h后转录量开始明显增加,24h时采用Western blot法能够检测到VP22蛋白,至72h～84h时UL49基因的转录量和表达量均达到最大值,之后开始逐渐降低。
5、鸭瘟病毒VP22蛋白核定位信号研究。为了研究DPV VP22蛋白是否存在核定位信号及其功能位点的位置,以绿色荧光蛋白为标记,构建了一系列不同区域截短VP22蛋白缺失突变体与GFP融合表达的真核质粒,然后使用脂质体介导转染鸭胚成纤维细胞,通过荧光显微镜观察分析各个缺失突变体的亚细胞定位,从而确定与核定位相关的结构域,为进一步深入研究DPV VP22蛋白的结构与功能奠定了基础。试验结果表明：(1)DPV VP22蛋白具有核定位功能,并且这种功能不依赖其他DPV蛋白；(2)DPV VP22蛋白C端165aa～253aa区域能够独立作为核定位信号；(3)DPV VP22蛋白的核定位功能在活细胞及不同固定剂处理后的细胞中都能够检测到,所以其核定位功能不受固定剂的影响。
6、DPV UL49基因RNA干扰有效靶点筛选及其对病毒重要糖蛋白基因转录的影响。根据实验室提供的DPV UL49基因序列,由上海吉玛制药技术有限公司设计4个不同的shRNA序列,然后插入到pGPU6载体中。将4个RNAi真核质粒与第六章构建的VP22a-GFP荧光质粒共转染鸭胚成纤维细胞,荧光显微镜观察干扰结果,4个RNAi干扰质粒均能够抑制VP22a-GFP质粒表达荧光,其中VP22-505和VP22-555表现的抑制作用最强。4种RNAi质粒对DPV UL49基因转录具有不同程度的抑制作用,尤其以VP22-505抑制效率最高,达到80%,从而确定了UL49基因中的“5-GCTGTTGGAGATTGCCAATGA-3"序列(即505bp-525bp)是RNAi发挥作用的最佳靶点。RNAi质粒VP22-505对DPV UL49基因转录抑制的同时,对DPV糖蛋白gB、gD和gE基因的转录也具有很强的抑制作用。
1. Bioinformatic analysis of UL27gene of Duck Plague Virus. In this paper, we conducted a detailed bioinformatic analysis of VP22protein. Our results showing that the DPV VP22protein has a molecular weight of27864.575Da, its pHi is computed to be9.795. The contents of Arg, Ala Ser, Asp and Thr were11.85%,11.85%,11.06%,7.11%and7.11%, respectively. For DPV VP22protein, the carboxyl terminus (126aa～191aa) is highly conserved. Similarity comparison and phylogenetic analysis showed that DPV aligns into the Alphaherpesvirus subfamily and is closely related to MDV. Two N-glycosylation sites, three cAMP-and cGMP-dependent protein kinase phosphorylation sites, nine protein kinase C phosphorylation sites, three Casein kinase II phosphorylation sites, two N-myristoylation sites, three Amidation sites were determined by PROSCAN. YLoc predicted that the DPV VP22protein sequence is located in the nucleus, cytoplasm and mitochondria with a probability of87.2%,12.7%and0.1%, respectively. Our results may be useful in future studies to elucidate the biological functions of VP22during DPV infection.
2. Cloning, prokaryotic expression, purification and polyclonal antibody preparation of DPV UL49gene. The full-length UL49gene of DPV was amplified with a pair of primer. This product was cloned into the multiple cloning site of the pMD19-T vector. For recombinant protein production, a prokaryotic expression vector pET28a(+) was used. pMD19-UL49was digested with BamH Ⅰ and Hind Ⅲ and the UL49sequense was inserted into the BamH Ⅰ/Hind Ⅲ site of pET-28a(+), which is capable of producing recombinant protein with an N-terminal6xHis tag. The result was designated pET28a-UL49and transformed into E. coli BL21(DE3) for recombinant protein production by calcium chloride method. For optimizing protein expression, cells were harvested4h after induction by0.6mM IPTG at37℃may prove optimal for the yield of soluble protein. For the production of the polyclonal rabbit anti-VP22serum, a adult New Zealand white rabbit was inoculated four times with VP22protein. The rabbit was bled7to10days after the final booster, then it was killed and the IgG fraction of the serum was isolated by ammonium sulfate precipitation. Then, using a DEAE-Sepharose column, the IgG fraction was purified by ion-exchange column chromatography. These results will provide a basis for further analysis of the DPV VP22protein. Western blots shows that the rabbit anti-VP22polyclonal antibody has good specificity.
3. Subcellular localization of Duck Plague Virus protein VP22. Indirect immunofluorescence tests revealed that the VP22antigens is detectable as early as6h postinfection, it exists predominantly in the cytoplasm and perinuclear region of DPV infected DEF in a diffuse fluorescent pattern early in infection, then migrates to and accumulates in the nucleus at12hpi, virtually every cell in the culture exhibited bright nucleus fluorescence at36hpi. At late stages of infection, cells expressing fluorescence dramatically reduced due to plaque famation at48hpi and72hpi. VP22found in infected cells is distributed in at least three distinct subcellular localizations, which we define as cytoplasmic, diffuse, and nuclear. Results of immunofluorescence studies show that the VP22recombinant protein can be detected in DEF after4h incubation in MEM, exhibiting pronounced accumulation in the cytoplasm, but failed migrates to and accumulates in the nucleus, virtually every cell in the culture exhibited bright cytoplasm fluorescence at36h. These results indicate that purified VP22recombinant protein retains the import property.
4. Transcription characteristics and expression kinetics analysis of Duck Plague Virus UL49gene. In this study, we determined the transcription characteristics and expression kinetics of DPV UL49gene by FQ-PCR and Western blot method. The results showed that DPV UL49gene transcripts appeared at1hpi, the relative expression level was at a low level in the first12hpi, and VP22protein can be detected at24hpi, and then the transcripts and VP22protein were peaked at72-84hpi, thereafter both of them declined.
5. Identification of the nuclear localization signal of Duck Plague Virus VP22protein. In order to mapped the functions of DPV VP22to specific regions in the polypeptide, we constructed a series of deletion constructs of VP22tagged by the GFP. The DPV VP22derivatives was transfected into DEF cells as described in Materials and Methods, and the subcellular localization pattern of the fusion proteins were examined using a Nikon Eclipse TE2000-U Inverted Microscope or a Nikon ECLIPSE80i fluorescence microscope. In this report, we demonstrate for the first time that (1) the nuclear localization of VP22is independent of other viral proteins,(2) VP22does not have a classical NLS, but a89-amino acid sequence from165aa to253aa within the carboxyl terminus was sufficient for nuclear localization.(3) the nuclear localization of VP22is independent of fixing agent.
6. The determination of RNA interference target for DPV UL49gene and its transcriptive effect to glycoprotein genes. According to the DPV UL49gene sequence,4shRNA were designed and constructed in pGPU6plasmids by Shanghai GenePharma Co.,Ltd.4RNAi plasmids and VP22a-GFP were cotransfected in DEF cells using LipofectamineTM2000Reagent, the results showed that VP22-505and VP22-555have inhibitive effect. Relative quantitative real-time RT-PCR analysis of UL49mRNA levels throughout DPV infections showed the "5-GCTGTTGGAGATTGCCAATGA-3" sequence (505bp～525bp) can be seen as the best target spot. When DEF cells were transfected with VP22-505expression vectors and then infected with DPV, the transcriptional effect of UL49, gB, gD and gE can be severely down-regulated by VP22-505.
引文
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