本文選題：豬博卡病毒 + 重組桿狀病毒��； 參考：《山東大學(xué)》2016年博士論文

【摘要】：豬博卡病毒(Porcine Bocavirus, PBoV),是細(xì)小病毒科(Family Parvoviridae)、細(xì)小病毒亞科(Subfamily Parvoviridae)、博卡病毒屬(Bocavirus Genus)成員之一,最早在2009年,由瑞典科學(xué)家,在患有離乳后、多系統(tǒng)衰竭綜合癥(Post-weaning multisystemic wasting syndrome, PMWS)的豬淋巴結(jié)中發(fā)現(xiàn),之后,世界各國(guó)也陸續(xù)發(fā)現(xiàn)了此種病毒。到現(xiàn)在為止,PBoV感染已在瑞典、中國(guó)、美國(guó)、加拿大、墨西哥、羅馬尼亞、匈牙利、烏干達(dá)、韓國(guó)、日本等國(guó)家均有報(bào)道。PBoV是無(wú)包膜、二十面體對(duì)稱(chēng)、單鏈DNA病毒。病毒顆粒的直徑25-30nm。基因組全長(zhǎng)約5000bp,包含3個(gè)主要開(kāi)放讀碼框架(open reading frames, ORFs), ORF1、ORF2、ORF3。ORF1,位于基因組5’端,編碼非結(jié)構(gòu)蛋白NS1。ORF1含有保守序列,與病毒的滾環(huán)復(fù)制、解旋酶、以及ATP酶活性有關(guān)。ORF2,位于基因組3’端,編碼兩個(gè)主要的衣殼蛋白VP1、VP2,參與病毒顆粒的組裝,決定抗原性。ORF3編碼功能未知的非結(jié)構(gòu)蛋白NP1,研究報(bào)道此蛋白是犬微小病毒(Canine minute virus, CMV)復(fù)制的必需蛋白。也有報(bào)道HBoV的NP1基因,可以阻斷干擾素(IFN)的合成,可能參與免疫逃逸。PBoV的分類(lèi)方法還沒(méi)有統(tǒng)一,按照VP1基因核苷酸序列的不同,PBoV可分為三個(gè)基因組(Groups): PBoV G1、PBoV G2,和PBoV G3。其中PBoV G3又可以分成5個(gè)亞基因組(Subgroups)。到目前為止,關(guān)于PBoV的致病性的分析研究數(shù)據(jù)有限,但PMWS患病豬的PBoV的血清陽(yáng)性率明顯高于非PMWS豬群,說(shuō)明PBoV可能與PMWS相關(guān)。另外,PBoV常與豬圓環(huán)病毒(Porcine circovirus, PCV)、豬細(xì)環(huán)病毒(Porcine torque teno virus, PTTV)、豬流行性腹瀉病毒(Porcine epidemic diarrhea virus, PEDV)等共感染,PBoV在淋巴結(jié)、血清、腸道等許多組織中被檢測(cè)到,說(shuō)明病毒可能會(huì)引起其它方面的病變。目前,對(duì)單一PBoV的發(fā)病機(jī)理,還沒(méi)有明確的證據(jù)。在病毒檢測(cè)方面,目前已經(jīng)建立了幾種不同的PCR和定量PCR的方法,用于檢測(cè)病毒核酸。同時(shí)也可用間接免疫熒光檢測(cè)法、以及酶聯(lián)免疫法(Enzyme-linked Immuno Sorbent Assay, ELISA),來(lái)檢測(cè)病毒抗原。但由于缺乏特異性好、敏感性高的抗-PBoV抗體的檢測(cè)方法,大規(guī)模的PBoV流行病學(xué)調(diào)查尚未能很好的開(kāi)展。因此,通過(guò)基因工程方法表達(dá)病毒抗原來(lái)檢測(cè)抗體,對(duì)研究病毒的抗原性和免疫原性、進(jìn)而對(duì)于開(kāi)展PBoV的流行病學(xué)調(diào)查、乃至病毒疫苗的研制都有重要意義。目前病毒蛋白可以通過(guò)細(xì)菌、酵母、昆蟲(chóng)細(xì)胞、哺乳類(lèi)動(dòng)物細(xì)胞等多種表達(dá)系統(tǒng)來(lái)表達(dá)。細(xì)菌(主要是大腸桿菌)表達(dá)系統(tǒng),有操作簡(jiǎn)單、產(chǎn)量高的優(yōu)點(diǎn),但不能對(duì)蛋白進(jìn)行糖基化修飾；酵母表達(dá)系統(tǒng),其產(chǎn)物與天然分子也還有一定差異；哺乳動(dòng)物表達(dá)系統(tǒng),雖然產(chǎn)物接近天然蛋白,但產(chǎn)量很低。昆蟲(chóng)細(xì)胞表達(dá)系統(tǒng),作為真核細(xì)胞(Eukaryocyte)表達(dá)系統(tǒng),表達(dá)產(chǎn)量非常高,產(chǎn)物的功能與結(jié)構(gòu)與天然蛋白相近。在昆蟲(chóng)細(xì)胞表達(dá)系統(tǒng)中,桿狀病毒(Baculovirus)表達(dá)系統(tǒng)已被廣泛應(yīng)用于類(lèi)病毒顆粒(Virus-like particles)的合成。其工作原理是：將外源目的基因,通過(guò)基因重組(Gene recombination)的方式,整合到多角體病毒(AcNPV)的多角體蛋白基因的啟動(dòng)子下游,然后將重組的桿狀病毒(recombinant baculorirus)感染昆蟲(chóng)細(xì)胞；當(dāng)Recombinant baculovirus在昆蟲(chóng)細(xì)胞內(nèi)復(fù)制時(shí),外源目的基因,可以利用多角體蛋白強(qiáng)悍的啟動(dòng)子的啟動(dòng)作用,在昆蟲(chóng)細(xì)胞中得到大量表達(dá)。產(chǎn)生的目的蛋白,具備與天然蛋白質(zhì)相類(lèi)似的功能和結(jié)構(gòu),抗原性也非常接近。特別是在表達(dá)無(wú)包膜病毒的構(gòu)造蛋白時(shí),可以在昆蟲(chóng)細(xì)胞內(nèi),自動(dòng)裝配成類(lèi)病毒顆粒(VLPs)。這種VLPs,不僅在結(jié)構(gòu)上與原始病毒的結(jié)構(gòu)相近,而且容易純化,可以獲得高產(chǎn)量、高純度的抗原,適用于血清抗體的檢測(cè),以及病毒疫苗的開(kāi)發(fā)研制。目的利用桿狀病毒表達(dá)系統(tǒng)(Baculovirus expressing system),在昆蟲(chóng)Tn5細(xì)胞中表達(dá)PBoV的主要構(gòu)造蛋白VP2,以期獲得PBoV的類(lèi)病毒顆粒(PBoV-LPs)。以PBoV-LPs為抗原,建立酶聯(lián)免疫法(ELISA),檢測(cè)抗-PBoV抗體,進(jìn)而探討PBoV的血清流行病學(xué)的特征。同時(shí)研究PBoV的免疫原性及免疫反應(yīng)性,為PBoV疫苗的研制開(kāi)發(fā)奠定基礎(chǔ)。也為未知病毒的抗原性的研究建立一套研究體系和研究模型。方法1.運(yùn)用PCR法,擴(kuò)增PBoV的VP2基因,將VP2基因克隆到TA克隆載體,并進(jìn)行大量擴(kuò)增。2.構(gòu)建VP2基因轉(zhuǎn)移載體pVL1393-VP2,與線性化的桿狀病毒DNA,一起轉(zhuǎn)染昆蟲(chóng)細(xì)胞Sf9,得到重組桿狀病毒Ac[VP2]。之后,在Sf9細(xì)胞中,使其大量擴(kuò)增。3.將重組桿狀病毒Ac[VP2],感染BTL-Tn5B1-4 (Tn5)細(xì)胞,進(jìn)行蛋白表達(dá)。動(dòng)態(tài)觀察蛋白表達(dá)：將細(xì)胞內(nèi)的蛋白、上清中的蛋白進(jìn)行SDS-PAGE電泳,考馬氏亮藍(lán)(Coomassie blue)染色后,觀察細(xì)胞內(nèi)蛋白的表達(dá),以及培養(yǎng)上清中蛋白的分泌,以確定回收蛋白的時(shí)間。4.收集培養(yǎng)7天后細(xì)胞上清液,離心、4℃重懸過(guò)夜后,進(jìn)行CsCl密度梯度離心純化蛋白。用蛋白測(cè)序方法,檢測(cè)蛋白質(zhì)N端氨基酸序列,驗(yàn)證VP2蛋白的準(zhǔn)確性。Uranyl acetate染色后,電鏡下觀察類(lèi)病毒顆粒。5.將PBoV-LPs免疫家兔,獲取抗PBoV-LPs抗體。利用該抗體和其它病毒顆粒的免疫反應(yīng),來(lái)確認(rèn)PBoV-LPs與HBoVs、以及PCV2-LPs的交叉反應(yīng)性。6.建立抗體檢測(cè)的方法,通過(guò)檢測(cè)家豬和野豬血清中抗PBoV IgG抗體,了解PBoV的流行病學(xué)狀況。7.檢測(cè)家豬和野豬血清PBoV的DNA,明確家豬和野豬中流行的PBoV的分子生物學(xué)特征。結(jié)果1.重組桿狀病毒AC[VP2]感染的Tn5細(xì)胞中,檢出PBoV VP2蛋白,其分子量約為61.5KDa,細(xì)胞內(nèi)蛋白的表達(dá)高峰,在感染后第三天到第五天,之后開(kāi)始遞減；上清液中的蛋白,在感染后第三天開(kāi)始檢測(cè)到,之后逐漸增多；感染后第7天,上清液中蛋白量達(dá)到高峰。2.感染后第七天收集細(xì)胞培養(yǎng)上清液,CsCl密度梯度離心法純化蛋白。分子量約為61.5KDa的蛋白質(zhì)集中在收集管8,9,10,密度為1.300g/cm3。電鏡下觀察到直徑約為30nm的類(lèi)病毒顆粒,形狀和原始病毒相似。蛋白質(zhì)序列分析顯示,類(lèi)病毒顆粒的N端5個(gè)氨基酸序列,與PBoV VP2蛋白的序列完全一致。說(shuō)明VLPs是由PBoV VP2基因表達(dá)而來(lái),而且PBoV VP2蛋白可以自行裝配成類(lèi)病毒顆粒(VLPs)。3.為驗(yàn)證病毒顆粒內(nèi)部是否包被了核酸,利用MagNA Pure LC Total NucleicAcid Isolation Kit (Roche Applied Science)試劑盒,從類(lèi)病毒顆粒中提取核酸,然后將提取物進(jìn)行瓊脂糖凝膠電泳,進(jìn)行觀察。結(jié)果顯示顆粒內(nèi)部未檢測(cè)到核酸。4.將病毒顆粒免疫家兔,進(jìn)行ELISA試驗(yàn),檢測(cè)抗體的效價(jià)。比較免疫前后兔血清中的抗體,免疫前兔血清中未檢出抗體；免疫后的兔血清中,抗PBoV-LPsIgG抗體的滴度高達(dá)1：409,600,說(shuō)明PBoV-LPs具有良好的免疫原性。5. PBoV與HBoVs的抗原性比較：通過(guò)ELISA法檢測(cè)抗PBoV-LPs抗體與HBoV-LPs、抗HBoV-LPs抗體與PBoV-LPs的交叉反應(yīng)性,來(lái)比較PBoV與HBoVs的抗原性的差別。發(fā)現(xiàn)兔抗-PBoV IgG與HBoV1-LPs和HBoV2-LPs均反應(yīng),但不與HBoV3-LPs和HBoV4-LPs反應(yīng)。而PBoV-LPs,可與兔抗-HBoV1,2,3,4-LPs IgG發(fā)生交叉反應(yīng),但弱于同源抗原抗體間的反應(yīng)。說(shuō)明PBoV可與HBoVs之間有相同的抗原決定簇存在。PBoV-LPs不與兔抗-PCV2-LPs IgG反應(yīng),PCV2-LPs也不與兔抗-PBoV-LPs IgG發(fā)生反應(yīng),說(shuō)明PBoV與PCV2抗原性完全不同。6.家豬和野豬的抗PBoV IgG抗體攜帶率：以PBoV-LPs作為抗原進(jìn)行ELISA試驗(yàn)檢測(cè)抗體。檢測(cè)了194份家豬和259份野豬血清,其結(jié)果,家豬血清抗體陽(yáng)性率高于90.7%(176/194),野豬血清抗體陽(yáng)性率平均為59.5%(154/259),證明PBoV的感染在家豬和野豬中是很普遍的現(xiàn)象。7. PBoV的病毒核酸檢測(cè)：利用巢式PCR擴(kuò)增PBoV NS1部分基因,調(diào)查家豬及野豬的病毒攜帶狀況。從259份野豬血清中共檢出7份血清PBoV DNA陽(yáng)性,其中兵庫(kù)縣采集的血清中有4份,茨城縣2份、長(zhǎng)崎縣1份。核苷酸序列分析結(jié)果表明,這7例病毒DNA屬于PBoV G3型基因組,并可以細(xì)分成4個(gè)亞型基因組,有明顯的地域區(qū)別。家豬血清樣本中,并未檢出病毒DNA,其原因在于,90%以上的家豬都已有抗體,病毒已被清除。結(jié)論1.本課題成功地利用桿狀病毒表達(dá)體系,合成了豬博卡病毒類(lèi)病毒顆粒(PBoV-LPs),并完成類(lèi)病毒顆粒的鑒定,包括蛋白的表達(dá)量、直徑、電鏡形態(tài)、分子量、顆粒密度、和免疫原性等生物學(xué)指標(biāo)。PBoV-LPs豬博卡病毒類(lèi)病毒顆粒具有天然VP2蛋白的特性及免疫原性,可作為抗原用于抗體的檢測(cè)以及疫苗的研制。2.應(yīng)用純化的PBoV-LPs作抗原,建立了抗體檢測(cè)的ELISA方法。并用此方法對(duì)PBoV的抗原性進(jìn)行了比較。發(fā)現(xiàn)PBoV可與HBoVs發(fā)生交叉反應(yīng),PBoV與PCV2的抗原性完全不同。3.對(duì)家豬和野豬的PBoV的感染狀況,進(jìn)行了流行病學(xué)調(diào)查：發(fā)現(xiàn)在家豬和野豬中,PBoV的感染都很普遍。家豬的感染率更高,和飼養(yǎng)環(huán)境有密切關(guān)聯(lián)。從野豬標(biāo)本中檢出的PBoV DNA序列分析表明,PBoV的感染存在多樣化、地域性差異等特點(diǎn)。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S852.65;Q78

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 楊蘭;卜志平;劉國(guó)敏;王曉鵬;;病毒的檢測(cè)方法[J];腦與神經(jīng)疾病雜志;2008年04期

2 洪濤,范瑞蓮,周靜儀,胥愛(ài)源,葉薇薇;人T細(xì)胞白血病病毒的電鏡研究[J];中國(guó)醫(yī)學(xué)科學(xué)院學(xué)報(bào);1987年02期

3 任向東;葛治華;余傳霖;胡新美;張惠星;高利;周紅;;巨細(xì)胞病毒中性紅斑定量試驗(yàn)與高滴度病毒的制備[J];病毒學(xué)雜志;1989年02期

4 毛江森,劉子陽(yáng),唐彩華,賀義惠,朱家鴻,王成玉,柴少愛(ài),陳悅青,錢(qián)汶;病毒顆粒中氫氘置換的證實(shí)[J];科學(xué)通報(bào);2004年01期

5 ;SARS研究重大進(jìn)展:空氣中病毒可被收集檢測(cè)[J];科技廣場(chǎng);2003年05期

6 常盛;;原始病毒顆粒[J];國(guó)外醫(yī)學(xué)情報(bào);1982年21期

7 何高魁，朱天成，王玉蘭;病毒快速自動(dòng)檢測(cè)法──一種新穎的核孔膜技術(shù)[J];物理;1995年02期

8 范宗理;;人類(lèi)H5N1型病毒的毒因[J];自然雜志;2006年04期

9 吳長(zhǎng)安;徐良;;病毒的寫(xiě)照:甲型流感病毒[J];國(guó)外醫(yī)學(xué)(微生物學(xué)分冊(cè));1979年03期

10 羅明典;;病毒應(yīng)用于農(nóng)業(yè)的一些進(jìn)展[J];病毒學(xué)雜志;1986年03期

相關(guān)會(huì)議論文 前8條

1 王占科;楊莉萍;祝仲珍;;病毒實(shí)驗(yàn)室診斷方法學(xué)評(píng)價(jià)和進(jìn)展[A];中華醫(yī)院管理學(xué)會(huì)第十屆全國(guó)醫(yī)院感染管理學(xué)術(shù)年會(huì)論文匯編[C];2003年

2 李巖;馬光輝;蘇志國(guó);;乙肝病毒表面抗原類(lèi)病毒顆粒在層析中的結(jié)構(gòu)變化[A];生物顆粒與粉體制備、應(yīng)用技術(shù)研討會(huì)論文集[C];2010年

3 毛江森;劉子陽(yáng);唐彩華;賀義惠;朱家鴻;王成玉;柴少愛(ài);陳悅青;錢(qián)汶;;病毒顆粒中氫氘置換的證實(shí)[A];浙江省醫(yī)學(xué)科學(xué)院建院55周年院慶論文專(zhuān)輯[C];2005年

4 張珍坤;史林啟;;病毒基水凝膠[A];2012年全國(guó)高分子材料科學(xué)與工程研討會(huì)學(xué)術(shù)論文集（上冊(cè)）[C];2012年

5 張新生;丘福禧;沈居仁;龍沛然;劉璣昌;曾子安;邊利婭;;在昆明地區(qū)病毒性腹瀉便中發(fā)現(xiàn)的病毒顆�！案珊Ｗ印币蜃覽A];第四次全國(guó)電子顯微學(xué)會(huì)議論文摘要集[C];1986年

6 劉暢;郭曉奎;;病毒在腫瘤治療當(dāng)中的作用[A];上海市預(yù)防醫(yī)學(xué)會(huì)第二屆學(xué)術(shù)年會(huì)論文匯編[C];2006年

7 王翠娥;李豫川;吳小紅;曹軍田;嚴(yán)格;李金鳳;司炳銀;于曼;秦鄂德;祝慶余;;感染細(xì)胞中SARS相關(guān)病毒的形態(tài)學(xué)觀察[A];中華醫(yī)學(xué)會(huì)系列雜志SARS研究論文集[C];2003年

8 劉曉波;Liu DX;;IBV病毒S蛋白上影響病毒介導(dǎo)細(xì)胞融合的氨基酸位點(diǎn)研究[A];2006中國(guó)微生物學(xué)會(huì)第九次全國(guó)會(huì)員代表大會(huì)暨學(xué)術(shù)年會(huì)論文摘要集[C];2006年

相關(guān)重要報(bào)紙文章 前9條

1 印高樂(lè);臺(tái)灣制成非典似病毒顆粒[N];醫(yī)藥經(jīng)濟(jì)報(bào);2004年

2 趙京玲;檢測(cè)空氣中非典病毒有新法[N];保健時(shí)報(bào);2003年

3 記者 毛黎;美首次觀察到艾滋病病毒顆粒細(xì)胞外形成過(guò)程[N];科技日?qǐng)?bào);2008年

4 姜平波 程守勤;SARS病毒收集檢測(cè)器研制成功[N];中國(guó)醫(yī)藥報(bào);2003年

5 記者 王燕寧 張曄 通訊員 姜平波;SARS病毒快速富集和檢測(cè)儀器問(wèn)世[N];科技日?qǐng)?bào);2003年

6 身體周刊記者 屠俊;能殺所有病毒的萬(wàn)能藥？[N];東方早報(bào);2011年

7 記者 張建列 通訊員 張鵬飛 馮春;病毒有蹤可循[N];廣東科技報(bào);2014年

8 江南煙雨;病毒是啥樣子[N];大眾衛(wèi)生報(bào);2005年

9 李藝;測(cè)定乙肝病毒核酸有何意義[N];中國(guó)醫(yī)藥報(bào);2003年

相關(guān)博士學(xué)位論文 前3條

1 張文靜;豬博卡病毒類(lèi)病毒顆粒的表達(dá)以及在流行病學(xué)方面的應(yīng)用[D];山東大學(xué);2016年

2 丁曉然;病毒顆粒介導(dǎo)的寡核苷酸靶向給藥技術(shù)研究[D];中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院;2013年

3 盧新亞;SARS CoV類(lèi)病毒顆粒免疫原性的研究與桿狀病毒做為活病毒疫苗載體的初步探索[D];中國(guó)科學(xué)院研究生院（武漢病毒研究所）;2007年

相關(guān)碩士學(xué)位論文 前5條

1 徐婧雯;腸道病毒71型類(lèi)病毒顆粒在昆蟲(chóng)細(xì)胞Sf9中的表達(dá)[D];北京協(xié)和醫(yī)學(xué)院;2011年

2 李嫣;食源性病毒高通量檢測(cè)方法的研究[D];南昌大學(xué);2014年

3 黃泓泰;兩種G20株CA16病毒顆粒的生物學(xué)特性的分析[D];北京協(xié)和醫(yī)學(xué)院;2013年

4 秦沖;一種用于A型鴨甲肝病毒檢測(cè)的類(lèi)病毒顆粒制備[D];華中農(nóng)業(yè)大學(xué);2013年

5 劉立鴻;新疆株HPV16的全基因組序列分析以及病毒顆粒在293T細(xì)胞中包裝的初步研究[D];新疆大學(xué);2008年

，

本文編號(hào)：1933041