【作者】 张晨飞；

【导师】 秦爱建；

【作者基本信息】 扬州大学 ， 预防兽医学， 2009， 博士

【摘要】 血清I型马立克氏病病毒(Marek’s disease virus serotype 1,MDV-1)的UL49基因和UL13基因分别和单纯疱疹病毒I型的UL49及UL13基因同源,各自编码大小约27.6kDa和57.1kDa大小的蛋白,在HSV-1中,这两种蛋白均为主要的被膜蛋白。VP22被发现在不存在其他病毒蛋白的情况下具备蛋白转导的功能,并且目前已经有很多研究表明VP22能够转运其它蛋白在细胞间扩散,使其在克服基因治疗和基因免疫的缺陷方面具有得天独厚的优势。而UL13作为病毒自身编码的丝/苏氨酸蛋白激酶,在病毒复制、组装、以及调节宿主细胞转录翻译方面发挥着重要作用。在HSV中,VP22是UL13的主要病毒蛋白底物,并且有研究发现VP22的磷酸化可能影响该蛋白的定位,而在MDV中还未有类似报道。目前关于MDV VP22及UL13的研究相对较少,究竟MDV VP22是否也能作为蛋白转运的工具?又有哪些蛋白能够被该蛋白转运?MDV VP22的转运功能是否能够增强机体针对目的蛋白的免疫应答水平?VP22的转导特性又如何?在MDV中,UL13是否能够磷酸化VP22?该磷酸化修饰对VP22又有何影响?这些都需要进一步的实验进行探索。本研究旨在通过对上述问题的探索,为VP22进一步走向应用打好基础。1. VP22转运异源蛋白的研究MDV VP22具有独立的蛋白转导功能,能够在细胞间高效转导,为进一步探索该蛋白作为蛋白转运工具的可行性,本研究构建了GFP、AIV-NP、BoIFN-γ、IBDV-VP2以及NDV-F基因与MDV-1 VP22融合表达的重组质粒,并将所获得的重组质粒在COS-1上进行瞬时表达以观察上述不同融合蛋白在COS-1细胞上的定位情况,从而评价VP22对这4种蛋白的转运能力。结果发现GFP、AIV-NP及BoIFN-γ在与VP22融合表达的状态下能够被VP22高效转运,而VP22对NDV-F的转运效率较低,对IBDV-VP2则完全不具备转运能力。此外,被VP22转运后的蛋白均定位在细胞核内。这些研究结果说明MDV VP22能够作为蛋白转运的载体,但对所转运蛋白具有选择性,并且VP22能够改变被转运蛋白原始的细胞定位。2. VP22增强机体针对目的抗原免疫应答水平的评价MDV VP22蛋白具备蛋白转运的功能,能够转运与VP22融合表达的GFP、AIV-NP、BoIFN-γ以及NDV-F等蛋白,为进一步评价VP22对这几种蛋白的免疫增强效果,本研究将pNP-VP22、pBoIFN-γ-VP22及pF-VP22的重组表达融合蛋白的质粒免疫Balb/c小鼠,4免后采取小鼠血清并分离小鼠淋巴细胞,通过ELISA、ELISPOT以及流式细胞法检测各组小鼠的免疫应答水平,结果发现,VP22能够特异性增强BoIFN-γ的细胞免疫水平,而对其体液免疫没有多大的影响。相反,NP蛋白的体液免疫水平却显著增强,但细胞免疫增强的效果不明显。而对于F蛋白,VP22几乎对其无任何的免疫增强的效果。上述结果显示,MDV-1 VP22具备增强机体针对目的抗原的免疫应答的功能,但免疫应答的类型可能会受到VP22蛋白转运方式的影响。3. VP22转导机制的研究MDV VP22蛋白具有独立的蛋白转导功能,能够作为蛋白转运的工具,但其转导及细胞定位的机制还未确定。我们早期的研究发现,在MDV感染的CEF中,VP22定位于细胞核,为进一步研究该蛋白的转导机制及细胞定位机理,分析该蛋白在表达过程中细胞定位的影响因素,本研究通过重组人腺病毒表达VP22蛋白,并对重组病毒表达的VP22的转导功能进行鉴定,结果发现将重组病毒感染的AD-293细胞裂解产物加至正常的MDBK细胞上,VP22能够进入几乎所有的细胞,说明重组病毒表达的VP22蛋白具有很强的蛋白转导功能。进一步鉴定VP22的细胞定位发现,在重组病毒感染的AD-293细胞中,VP22首先聚集于细胞核周围,随后以特殊的荧光粒子的形式散在于胞浆中,有别于AD-293细胞中瞬时表达的VP22及MDV感染的CEF中VP22的定位模式;同时我们也对Bac-to-Bac杆状病毒表达系统表达的VP22的蛋白转导特性以及VP22转导的细胞广谱性进行了研究,发现该系统表达的VP22也具有蛋白转导的功能,并且其定位可能受到温度的影响,而瞬时表达的VP22的转导是细胞广谱性的,能够在多种细胞上实现蛋白转导的功能,并且定位在细胞核内。4. MDV-1 UL13序列对比分析及对VP22可能的磷酸化位点预测蛋白激酶是一类庞大的蛋白家族,尽管它们的结构、催化模式以及特异性底物都存在很大的差异,但它们的功能结构域却相当保守。MDV UL13是病毒编码的蛋白激酶,本研究通过DNAStar软件的MegAlin功能对比MDV不同毒株以及不同疱疹病毒属的UL13氨基酸序列,发现MDV不同毒株的UL13序列近乎相同,而不同疱疹病毒属的UL13及相应同源物的同源性很低,但在它们的激酶结构域内保守。通过CDTree软件绘制该蛋白的遗传分类图谱,发现它与黑腹果蝇的pelle蛋白的功能结构域属于相同进化分支,利用NCBI protein Blast功能检索MDV UL13保守结构域,发现MDV UL13也具有丝/苏氨酸蛋白激酶的激酶结构域,并且催化中心主要位于152-297氨基酸残基间,利用Cn3D 4.1软件分析Blast结果,建立UL13可能的结构模型,同时对比真核生物蛋白激酶的基序,发现UL13在激酶SubdomainⅦ的保守甘氨酸残基被丝氨酸替代,SubdomainⅧ的保守非极性脯氨酸残基被极性半胱氨酸残基替换。利用NetPhos 2.0 Server对VP22进行磷酸化位点预测,发现该蛋白存在多个丝/苏氨酸磷酸化位点,且主要集中在两端,提示MDV VP22很有可能也是UL13的磷酸化底物。5. UL13的原核和真核表达以及多抗血清的制备MDV UL13蛋白是病毒自身编码的蛋白激酶,具有类似真核细胞蛋白激酶的功能,并且在整个疱疹病毒科内都具有保守性,为研究该蛋白激酶的功能,本研究通过PCR方法从CVI988疫苗株基因组中扩增UL13基因,并将UL13基因片段克隆到杆状病毒转移载体pFastTMBac1中,再将重组质粒转化DH10Bac感受态细胞,经过转座和蓝白菌落筛选及PCR鉴定,获得含UL13基因的重组穿梭载体。在脂质体的辅助下将重组穿梭载体转染Sf9细胞,通过PCR验证获得含UL13基因的重组杆状病毒,命名为rBac-UL13。同时,利用GENEART(www.gcua.de)分析UL13在大肠杆菌中表达时密码子的偏嗜性;通过DNAstar抗原性分析确定UL13的高抗原性片段,进行原核表达,并以切胶免疫方法免疫小鼠制备多抗血清,再以获得的多抗血清检测rBac-UL13重组毒感染的sf9细胞,证实了所获多抗血清含有特异性针对UL13的抗体,同时也证实了UL13在Bac-to-Bac杆状病毒表达系统中的表达。更多还原

【Abstract】 The UL49 and UL13 homolog gene of Herpes simplex virus (HSV) in Marek’s disease virus serotype 1 (MDV-1) encodes the major tegument protein VP22 and UL13 of 27.6kDa and 57.1kDa respectively. VP22 possess a remarkable property of protein transduction independent of any other viral proteins and can be used in protein delivery technology to overcome the limitations in gene therapy and DNA vaccine. UL13 plays important roles in virus replication, virion assembly, regulation of host cells’transcription and translation as a viral serine/threonine protein kinase. VP22 is a major substrate of UL13 in HSV, and phosphorylation of VP22 may influence the localization of this protein. However, phosphorylation of VP22 by UL13 in MDV has not been demonstrated so far.Currently, most researches about VP22 and UL13 are focus on HSV. However, whether can MDV VP22 be utilized as a transporter for protein delivery? If Yes, what kinds of proteins can be transported by MDV VP22? Can MDV VP22 enhance the immune response induced by these proteins? What is the transduction characterization of MDV VP22? Does UL13 phosphorylate VP22 in MDV? Does the modification of phosphorylation influence the biological activity of VP22? All these questions remain unclear. We aimed to resolve these questions in this study for the further application of MDV VP22.1. MDV-1 VP22: a transporter that can selectively deliver proteins into cellsMDV VP22 shows a remarkable property of protein transduction independent of any other viral proteins. To confirm whether it can be utilized as a transporter for protein delivery, five heterogenes, encodes GFP, AIV-NP, BoIFN-γ, IBDV-VP2 and NDV-F were respectively fused to MDV UL49 gene. Transient expression were carried out by transfecting these fusion genes into COS-1 cells. The results suggested that GFP, AIV-NP、BoIFN-γbe highly transported by VP22 while NDV-F be transported at low efficiency. However IBDV-VP2 could not be deliveried at all. Our data suggests that the VP22 of MDV-1 selectively transport heterogenous proteins into cells and the original localization of cargo proteins may be changed after transporting by MDV-1 VP22.2. Evaluation of MDV VP22 enhances the immune response by DNA vaccinationMDV VP22 possesses the property of protein delivery and GFP, AIV-NP, BoIFN-γ, IBDV-VP2 and NDV-F can be deliveried into cells by VP22. To further evaluate the immune response enhancements induced by VP22, balb/c mice were immunized with recombinant plasmids expressing NP-VP22, BoIFN-γ-VP22 and F-VP22 respectively. Antiserum and spleen cells were isolated from the mice after 4 times immunization. ELISA, ELISPOT and FACS analysis was carried out to evaluate the immune response of each group. The results indicated that the cellular immunity level of BoIFN-γwas significantly enhanced by VP22 while the humoral immunity was not. In contrast, humoral immunity level of NP was significantly enhanced and a slight enhancement of cellular immunity. However, the immune response induced by F-VP22 and F alone has no difference. It implied that the immunogenicity of F can not be enhanced by MDV VP22. Taken together, MDV-1 VP22 selectively increases the immunity of antigens, but the type of immune response may be influenced by the way in which VP22 transported.3. Mechanism of VP22 protein transductionVP22 shows a remarkable property of protein transduction independent of any other viral proteins, and can be used for protein delivery. However, the subcellular localization mechanism of this protein remains unclear. In this study, VP22 was expressed with recombinant adenovirus. Lysates of recombinant virus infected 293 cells were added to normal MDBK cells. The results revealed that infected 293 cells expressed VP22 protein can almost enter all the monolayers, suggests that adenovirus expressed VP22 remains its transduction property. Subsequent study showed that in recombinant adenovirus infected 293 cells, VP22 first gather round the nucleus membrane, and then concentrated in particles in cytoplasm, which differs from the nuclei localization pattern of VP22 in MDV infected CEF and transient expressed VP22 in 293 cells. We also characterized the protein transduction property of Bac-to-Bac Baculovirus Expression System expressed VP22, and examined the transduction ability of transient expressed VP22 in different cell lines. The results suggested that VP22 expressed by Bac-to-Bac System remained its protein transduction property and the subcellular localization of the protein may be influenced by temperature; transient expressed VP22 remained its transduction property in different cells and was localized in the nucleolus of all these cell types.4. Sequence analysis of MDV-1 UL13 and phosphorylation sites prediction of VP22The protein kinases comprise one of the largest superfamilies of homologous proteins containing hundreds of members. Although there is a rich diversity of structures, regulation modes, and substrate specificities among the protein kinases, there are also common motifs and structural features in their functional domains. In this study, amino acid sequence analysis was carried out between the homologous gene of UL13 in different strains of MDV and in different Herpesviridae subfamilies, analysis by MegAlin function of DNAstar software. The results indicted that UL13 share almost 100% homology between different strains of MDV, and was highly conserved in kinase domains while the sequences contained a rich diversity in different Herpesviridae subfamilies. Taxonomy analysis of UL13 catalytic center using CDTree3.1 software provided by NCBI implied that UL13 is more homologous to Drosophila melanogaster pelle protein. The conserved domain of UL13 was analyzed with protein blast and Cn3D 4.1 online software of NCBI, the results suggest that 152-297 residue is kinase catalytic center of UL13. However conserved glycine in kinase subdomainⅦfor most protein kinase was replaced by serine in UL13 and proline in kinase subdomainⅧreplaced by cysteine. Phosphorylation sites prediction of VP22 was carried out with NetPhos 2.0 online Server. Numerous phosphorylation sites were found, most of which were enriched in the N and C terminus of VP22, implying that VP22 may be a kinase substrate of UL13 in MDV.5. Expression of UL13 by prokaryotic and eukaryotic system and antiserum preparationAs a viral serine/threonine protein kinase, the kinase activity of UL13 is similar to the eukaryotic protein kinases, and is conserved in Herpesviridae subfamily. To further investigate the function of UL13 kinase, UL13 gene was amplified by polymerase chain reaction (PCR) from MDV-1 CVI988/Rispens strain, followed by introducing to the pFastTMBac1 vector. The recombinants were transformed into DH10Bac in which translocation was occurred. Recombinant genome of baculovirus was identified by PCR and then transfected into sf9 cells to obtain the recombinant baculovirus expressing UL13, named as rBac-UL13. The codon bias and antigenicity of UL13 in E.coli was analyzed by online service GENEART ( www.gcua.de ) and DNAstar software respectively. The UL13 truncated fragments were expressed in E.coli as GST fusion protein, and mice were immunized with the expressed GST fusion protein to obtain antiserum against UL13. Sf9 cells infected with rBac-UL13 were reacted with the antiserum. The result suggested that the antiserum contains antibodies specific to UL13.更多还原