猪博卡病毒类病毒颗粒的表达以及在流行病学方面的应用

发布时间：2018-05-25 10:09

本文选题：猪博卡病毒 + 重组杆状病毒　；参考：《山东大学》2016年博士论文

【摘要】：猪博卡病毒(Porcine Bocavirus, PBoV),是细小病毒科(Family Parvoviridae)、细小病毒亚科(Subfamily Parvoviridae)、博卡病毒属(Bocavirus Genus)成员之一,最早在2009年,由瑞典科学家,在患有离乳后、多系统衰竭综合症(Post-weaning multisystemic wasting syndrome, PMWS)的猪淋巴结中发现,之后,世界各国也陆续发现了此种病毒。到现在为止,PBoV感染已在瑞典、中国、美国、加拿大、墨西哥、罗马尼亚、匈牙利、乌干达、韩国、日本等国家均有报道。PBoV是无包膜、二十面体对称、单链DNA病毒。病毒颗粒的直径25-30nm。基因组全长约5000bp,包含3个主要开放读码框架(open reading frames, ORFs), ORF1、ORF2、ORF3。ORF1,位于基因组5’端,编码非结构蛋白NS1。ORF1含有保守序列,与病毒的滚环复制、解旋酶、以及ATP酶活性有关。ORF2,位于基因组3’端,编码两个主要的衣壳蛋白VP1、VP2,参与病毒颗粒的组装,决定抗原性。ORF3编码功能未知的非结构蛋白NP1,研究报道此蛋白是犬微小病毒(Canine minute virus, CMV)复制的必需蛋白。也有报道HBoV的NP1基因,可以阻断干扰素(IFN)的合成,可能参与免疫逃逸。PBoV的分类方法还没有统一,按照VP1基因核苷酸序列的不同,PBoV可分为三个基因组(Groups): PBoV G1、PBoV G2,和PBoV G3。其中PBoV G3又可以分成5个亚基因组(Subgroups)。到目前为止,关于PBoV的致病性的分析研究数据有限,但PMWS患病猪的PBoV的血清阳性率明显高于非PMWS猪群,说明PBoV可能与PMWS相关。另外,PBoV常与猪圆环病毒(Porcine circovirus, PCV)、猪细环病毒(Porcine torque teno virus, PTTV)、猪流行性腹泻病毒(Porcine epidemic diarrhea virus, PEDV)等共感染,PBoV在淋巴结、血清、肠道等许多组织中被检测到,说明病毒可能会引起其它方面的病变。目前,对单一PBoV的发病机理,还没有明确的证据。在病毒检测方面,目前已经建立了几种不同的PCR和定量PCR的方法,用于检测病毒核酸。同时也可用间接免疫荧光检测法、以及酶联免疫法(Enzyme-linked Immuno Sorbent Assay, ELISA),来检测病毒抗原。但由于缺乏特异性好、敏感性高的抗-PBoV抗体的检测方法,大规模的PBoV流行病学调查尚未能很好的开展。因此,通过基因工程方法表达病毒抗原来检测抗体,对研究病毒的抗原性和免疫原性、进而对于开展PBoV的流行病学调查、乃至病毒疫苗的研制都有重要意义。目前病毒蛋白可以通过细菌、酵母、昆虫细胞、哺乳类动物细胞等多种表达系统来表达。细菌(主要是大肠杆菌)表达系统,有操作简单、产量高的优点,但不能对蛋白进行糖基化修饰；酵母表达系统,其产物与天然分子也还有一定差异；哺乳动物表达系统,虽然产物接近天然蛋白,但产量很低。昆虫细胞表达系统,作为真核细胞(Eukaryocyte)表达系统,表达产量非常高,产物的功能与结构与天然蛋白相近。在昆虫细胞表达系统中,杆状病毒(Baculovirus)表达系统已被广泛应用于类病毒颗粒(Virus-like particles)的合成。其工作原理是：将外源目的基因,通过基因重组(Gene recombination)的方式,整合到多角体病毒(AcNPV)的多角体蛋白基因的启动子下游,然后将重组的杆状病毒(recombinant baculorirus)感染昆虫细胞；当Recombinant baculovirus在昆虫细胞内复制时,外源目的基因,可以利用多角体蛋白强悍的启动子的启动作用,在昆虫细胞中得到大量表达。产生的目的蛋白,具备与天然蛋白质相类似的功能和结构,抗原性也非常接近。特别是在表达无包膜病毒的构造蛋白时,可以在昆虫细胞内,自动装配成类病毒颗粒(VLPs)。这种VLPs,不仅在结构上与原始病毒的结构相近,而且容易纯化,可以获得高产量、高纯度的抗原,适用于血清抗体的检测,以及病毒疫苗的开发研制。目的利用杆状病毒表达系统(Baculovirus expressing system),在昆虫Tn5细胞中表达PBoV的主要构造蛋白VP2,以期获得PBoV的类病毒颗粒(PBoV-LPs)。以PBoV-LPs为抗原,建立酶联免疫法(ELISA),检测抗-PBoV抗体,进而探讨PBoV的血清流行病学的特征。同时研究PBoV的免疫原性及免疫反应性,为PBoV疫苗的研制开发奠定基础。也为未知病毒的抗原性的研究建立一套研究体系和研究模型。方法1.运用PCR法,扩增PBoV的VP2基因,将VP2基因克隆到TA克隆载体,并进行大量扩增。2.构建VP2基因转移载体pVL1393-VP2,与线性化的杆状病毒DNA,一起转染昆虫细胞Sf9,得到重组杆状病毒Ac[VP2]。之后,在Sf9细胞中,使其大量扩增。3.将重组杆状病毒Ac[VP2],感染BTL-Tn5B1-4 (Tn5)细胞,进行蛋白表达。动态观察蛋白表达：将细胞内的蛋白、上清中的蛋白进行SDS-PAGE电泳,考马氏亮蓝(Coomassie blue)染色后,观察细胞内蛋白的表达,以及培养上清中蛋白的分泌,以确定回收蛋白的时间。4.收集培养7天后细胞上清液,离心、4℃重悬过夜后,进行CsCl密度梯度离心纯化蛋白。用蛋白测序方法,检测蛋白质N端氨基酸序列,验证VP2蛋白的准确性。Uranyl acetate染色后,电镜下观察类病毒颗粒。5.将PBoV-LPs免疫家兔,获取抗PBoV-LPs抗体。利用该抗体和其它病毒颗粒的免疫反应,来确认PBoV-LPs与HBoVs、以及PCV2-LPs的交叉反应性。6.建立抗体检测的方法,通过检测家猪和野猪血清中抗PBoV IgG抗体,了解PBoV的流行病学状况。7.检测家猪和野猪血清PBoV的DNA,明确家猪和野猪中流行的PBoV的分子生物学特征。结果1.重组杆状病毒AC[VP2]感染的Tn5细胞中,检出PBoV VP2蛋白,其分子量约为61.5KDa,细胞内蛋白的表达高峰,在感染后第三天到第五天,之后开始递减；上清液中的蛋白,在感染后第三天开始检测到,之后逐渐增多；感染后第7天,上清液中蛋白量达到高峰。2.感染后第七天收集细胞培养上清液,CsCl密度梯度离心法纯化蛋白。分子量约为61.5KDa的蛋白质集中在收集管8,9,10,密度为1.300g/cm3。电镜下观察到直径约为30nm的类病毒颗粒,形状和原始病毒相似。蛋白质序列分析显示,类病毒颗粒的N端5个氨基酸序列,与PBoV VP2蛋白的序列完全一致。说明VLPs是由PBoV VP2基因表达而来,而且PBoV VP2蛋白可以自行装配成类病毒颗粒(VLPs)。3.为验证病毒颗粒内部是否包被了核酸,利用MagNA Pure LC Total NucleicAcid Isolation Kit (Roche Applied Science)试剂盒,从类病毒颗粒中提取核酸,然后将提取物进行琼脂糖凝胶电泳,进行观察。结果显示颗粒内部未检测到核酸。4.将病毒颗粒免疫家兔,进行ELISA试验,检测抗体的效价。比较免疫前后兔血清中的抗体,免疫前兔血清中未检出抗体；免疫后的兔血清中,抗PBoV-LPsIgG抗体的滴度高达1：409,600,说明PBoV-LPs具有良好的免疫原性。5. PBoV与HBoVs的抗原性比较：通过ELISA法检测抗PBoV-LPs抗体与HBoV-LPs、抗HBoV-LPs抗体与PBoV-LPs的交叉反应性,来比较PBoV与HBoVs的抗原性的差别。发现兔抗-PBoV IgG与HBoV1-LPs和HBoV2-LPs均反应,但不与HBoV3-LPs和HBoV4-LPs反应。而PBoV-LPs,可与兔抗-HBoV1,2,3,4-LPs IgG发生交叉反应,但弱于同源抗原抗体间的反应。说明PBoV可与HBoVs之间有相同的抗原决定簇存在。PBoV-LPs不与兔抗-PCV2-LPs IgG反应,PCV2-LPs也不与兔抗-PBoV-LPs IgG发生反应,说明PBoV与PCV2抗原性完全不同。6.家猪和野猪的抗PBoV IgG抗体携带率：以PBoV-LPs作为抗原进行ELISA试验检测抗体。检测了194份家猪和259份野猪血清,其结果,家猪血清抗体阳性率高于90.7%(176/194),野猪血清抗体阳性率平均为59.5%(154/259),证明PBoV的感染在家猪和野猪中是很普遍的现象。7. PBoV的病毒核酸检测：利用巢式PCR扩增PBoV NS1部分基因,调查家猪及野猪的病毒携带状况。从259份野猪血清中共检出7份血清PBoV DNA阳性,其中兵库县采集的血清中有4份,茨城县2份、长崎县1份。核苷酸序列分析结果表明,这7例病毒DNA属于PBoV G3型基因组,并可以细分成4个亚型基因组,有明显的地域区别。家猪血清样本中,并未检出病毒DNA,其原因在于,90%以上的家猪都已有抗体,病毒已被清除。结论1.本课题成功地利用杆状病毒表达体系,合成了猪博卡病毒类病毒颗粒(PBoV-LPs),并完成类病毒颗粒的鉴定,包括蛋白的表达量、直径、电镜形态、分子量、颗粒密度、和免疫原性等生物学指标。PBoV-LPs猪博卡病毒类病毒颗粒具有天然VP2蛋白的特性及免疫原性,可作为抗原用于抗体的检测以及疫苗的研制。2.应用纯化的PBoV-LPs作抗原,建立了抗体检测的ELISA方法。并用此方法对PBoV的抗原性进行了比较。发现PBoV可与HBoVs发生交叉反应,PBoV与PCV2的抗原性完全不同。3.对家猪和野猪的PBoV的感染状况,进行了流行病学调查：发现在家猪和野猪中,PBoV的感染都很普遍。家猪的感染率更高,和饲养环境有密切关联。从野猪标本中检出的PBoV DNA序列分析表明,PBoV的感染存在多样化、地域性差异等特点。
[Abstract]:The porcine Boka virus (Porcine Bocavirus, PBoV) is one of the members of the parvovirus (Family Parvoviridae), parvovirus subfamily (Subfamily Parvoviridae) and the genus Boka virus (Bocavirus Genus). It was first in 2009 and was a Swedish scientist with multiple system failure syndrome (Post-weaning multisystemic wasting). So far, PBoV infection has been reported in Sweden, China, the United States, the United States, Canada, Mexico, Romania, Hungary, Uganda, Korea, Japan and other countries, which have reported that.PBoV is a non membrane, twenty body symmetry, single strand DNA virus. The diameter 25-30nm. of the virus particles. The total length of the genome is about 5000bp, including 3 main open reading code frames (open reading frames, ORFs), ORF1, ORF2, ORF3.ORF1, located at the 5 'end of the genome, and the encoded non structural protein NS1.ORF1 contains a conservative sequence, which is related to the viral roll ring replication, the helicase, and ATP enzyme activity.ORF2, located at the 3' end of the genome, and encodes two main capsid eggs. White VP1, VP2, participate in the assembly of virus particles, determine the unstructured protein NP1 of unknown.ORF3 encoding function of antigenicity. It is reported that this protein is a necessary protein for the replication of the canine parvovirus (Canine minute virus, CMV). It also reports the NP1 gene of HBoV, which can block the synthesis of interferon (IFN) and may be involved in the classification of immune escape.PBoV. No unification, according to the nucleotide sequence of the VP1 gene, PBoV can be divided into three genomes (Groups): PBoV G1, PBoV G2, and PBoV G3. of which PBoV G3 can be divided into 5 subgenomes (Subgroups). S pigs, indicating that PBoV may be associated with PMWS. In addition, PBoV is often associated with porcine circovirus (Porcine circovirus, PCV), porcine circovirus (Porcine Torque teno virus, PTTV), porcine epidemic diarrhea virus, and so on in many tissues such as lymph nodes, serum, intestine and so on. At present, there is no clear evidence for the pathogenesis of a single PBoV. In the field of virus detection, several different methods of PCR and quantitative PCR have been established to detect virus nucleic acid. Meanwhile, indirect immunofluorescence and Enzyme-linked Immuno Sorbent As are also used. Say, ELISA), to detect virus antigens. But because of the lack of specific and sensitive detection methods of anti -PBoV antibody, large-scale PBoV epidemiological investigation has not been well carried out. Therefore, the antigenicity and immunogenicity of the virus are studied by gene engineering method to study the antigenicity and immunogenicity of the virus, and then to carry out PBoV Epidemiological investigation and even the development of viral vaccines are of great significance. At present, viral proteins can be expressed through a variety of expression systems such as bacteria, yeast, insect cells and mammalian cells. The bacteria (mainly Escherichia coli) express system, which has the advantages of simple operation and high yield, but can not be glycosylated; yeast The expression system of the mammalian expression system, although the product is close to the natural protein, is very low in production. The expression system of the insect cell expression system, as the eukaryotic cell (Eukaryocyte) expression system, is very high in expression, and the function and structure of the product are similar to that of natural protein. The Baculovirus expression system has been widely used in the synthesis of Virus-like particles. The principle of this system is to integrate the exogenous target gene into the promoter of the polyhedrosis (AcNPV) polyhedron gene (AcNPV) gene by gene recombination (Gene recombination) and then recombine the recombinant baculovirus. Virus (recombinant baculorirus) infects insect cells; when Recombinant baculovirus replicates in insect cells, the exogenous target gene can be expressed in insect cells by the activation of the strong promoter of polyhedron protein. The target protein produced in the insect cells has the function and structure similar to that of the natural protein. The antigenicity is also very close. Especially in the expression of the structural protein without the envelope virus, it can be automatically assembled into the virus like particles (VLPs) in the insect cells. This VLPs is not only structurally similar to the structure of the original virus, but also easy to purify, and can obtain high yield, high purity antigen, suitable for detection of serum antibody and disease. The development and development of the virus vaccine. Aim to express the major structural protein VP2 of PBoV in the insect Tn5 cells (Baculovirus expressing system) in order to obtain the PBoV virus like particles (PBoV-LPs). The enzyme linked immunoassay (ELISA) was established with PBoV-LPs as the antigen, and the anti -PBoV antibody was detected. Then the serological epidemic of PBoV was discussed. At the same time, study the immunogenicity and immunoreactivity of PBoV, lay the foundation for the development and development of PBoV vaccine, and set up a set of research system and research model for the study of the antigenicity of the unknown virus. Method 1. the VP2 gene of PBoV was amplified by PCR method, the VP2 gene was cloned into the TA cloning vector, and a large number of.2. were amplified to construct VP2 base. The transfer vector pVL1393-VP2, transfected with the linearized baculovirus DNA, transfected the insect cell Sf9 together and obtained the recombinant baculovirus Ac[VP2]., in Sf9 cells, the recombinant baculovirus Ac[VP2], infected with BTL-Tn5B1-4 (Tn5) cells, were amplified by a large number of.3., and the protein expression was observed dynamically: the protein in the cell, the supernatant in the supernatant, was observed dynamically. After SDS-PAGE electrophoresis, the protein was stained with martensim blue (Coomassie blue), the protein expression in the cells and the secretion of protein in the supernatant were observed to determine the time for the recovery of protein..4. was collected and cultured for 7 days after the culture of the supernatant. After the centrifugation, the protein was purified by CsCl density gradient centrifugation. The protein was purified by CsCl density gradient, and the protein sequencing method was used. The sequence of protein N terminal amino acid was detected, and the accuracy of VP2 protein was verified by.Uranyl acetate staining. Under electron microscope, the virus like particle.5. was observed to immunize the rabbit and obtain anti PBoV-LPs antibody. The antibody and other virus particles were used to confirm the PBoV-LPs and HBoVs, and the PCV2-LPs cross reactivity.6. to establish the antibody detection. Methods to detect the anti PBoV IgG antibody in the serum of domestic pigs and wild boar, to understand the epidemiological status of PBoV and to detect the DNA of PBoV in domestic pigs and wild boar serum, and to identify the molecular biological characteristics of PBoV in domestic pigs and wild boars. Results 1. the PBoV VP2 protein was detected in Tn5 cells infected by the recombinant baculovirus AC[VP2], and the molecular weight of the protein was about 61.5KDa. The expression peak of intracellular protein, after infection third days to fifth days after infection, began to decrease, the protein in the supernatant began to be detected at third days after infection and then gradually increased; seventh days after infection, the protein content in the supernatant reached the peak.2. infection after seventh days to collect cell culture supernatant and CsCl density gradient centrifugation method. Protein. The protein with a molecular weight of about 61.5KDa is concentrated in the collection tube 8,9,10. The density of the virus particles with a diameter of about 30nm under the 1.300g/cm3. electron microscope is similar to that of the original virus. The protein sequence analysis shows that the 5 amino acid sequence of the N terminal of the virus like particles is exactly the same as the sequence of PBoV VP2 protein. It shows that VLPs is PBoV VP. The 2 gene was expressed, and the PBoV VP2 protein could be assembled into a virus like particle (VLPs).3. to verify whether the virus particles were wrapped in the nucleic acid. The nucleic acid was extracted from the virus like particles by using the MagNA Pure LC Total NucleicAcid Isolation Kit (Roche) reagent box, and then the extract was carried out by agarose gel electrophoresis. The results showed that the virus particles were not detected by nucleic acid.4. in the particle, and the virus particles were immune to the rabbit. The ELISA test was carried out to detect the titer of the antibody. The antibody in the rabbit serum before and after immunization was compared. The antibody in the rabbit serum before immunization was not detected. The titer of anti PBoV-LPsIgG anti body was up to 1:409600 in the rabbit serum after the immunization, indicating that the PBoV-LPs has a PBoV-LPs. The antigenicity of good immunogenicity.5. PBoV and HBoVs: the cross reactivity of anti PBoV-LPs antibody and HBoV-LPs, anti HBoV-LPs antibody and PBoV-LPs by ELISA method, to compare the difference between PBoV and HBoVs. S can cross reaction with Rabbit anti -HBoV1,2,3,4-LPs IgG, but it is weaker than the reaction between homologous antigen antibody. It shows that PBoV can have the same antigenic determinant as HBoVs,.PBoV-LPs does not react with Rabbit anti -PCV2-LPs IgG, PCV2-LPs also does not react with Rabbit anti -PBoV-LPs IgG, saying that PBoV and antigenicity are completely different from swine and wild boar. The anti PBoV IgG antibody carrying rate: using PBoV-LPs as the antigen to test the antibody by ELISA test. 194 domestic pigs and 259 Bleno Choshi were detected. The results showed that the positive rate of serum antibody of domestic pigs was higher than 90.7% (176/194), and the positive rate of serum antibody of wild boar was 59.5% (154/259). It proved that the infection of PBoV was a common phenomenon in domestic pigs and wild boars. 7. PBoV virus nucleic acid detection: using the nested PCR to amplify the PBoV NS1 gene and investigate the virus carrying status of domestic pigs and wild boar. 7 serum PBoV DNA positive were detected from 259 wild boar serum, of which there were 4 serums in Hyogo County, 2 in Ibaraki county and 1 in Ibaraki county. The nucleotide sequence analysis showed that these 7 virus DNA belonged to the sera. The PBoV G3 genomes can be subdivided into 4 subtypes, and there are obvious regional differences. The virus DNA is not detected in the pig serum samples. The reason is that more than 90% of the domestic pigs have antibodies and the virus has been cleared. Conclusion the 1. subjects successfully used baculovirus system to synthesize the porcine Boka virus particles (PBoV-L Ps) and complete identification of virus like particles, including protein expression, diameter, electron microscope morphology, molecular weight, particle density, and immunogenicity, and other biological indicators.PBoV-LPs porcine Boka virus particles have the characteristics and immunogenicity of natural VP2 protein, which can be used as Antiogen for antibody detection and vaccine development and.2. application and purification. PBoV-LPs was used as an antigen and a ELISA method for antibody detection was established. The antigenicity of PBoV was compared by this method. It was found that PBoV could cross reaction with HBoVs. The antigenicity of PBoV and PCV2 was completely different from.3. to PBoV in domestic pigs and wild boars. Epidemiological investigation was carried out: the infection of PBoV was found in domestic pigs and wild boars. It is common that the infection rate of domestic pigs is higher and closely related to the feeding environment. The analysis of PBoV DNA sequence detected from the specimens of wild boar shows that the infection of PBoV has the characteristics of diversity, regional difference and so on.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65;Q78

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