【作者】 周斌；

【导师】 陈溥言；

【作者基本信息】 南京农业大学 ， 生物技术， 2010， 博士

【摘要】 猪瘟(Classical Swine Fever, CSF)是一种由猪瘟病毒(Classical Swine Fever virus, CSFV)引起的急性、热性和高度接触性的传染病,流行广泛,发病率高、死亡率高,危害极大,给世界和我国的养猪业造成严重经济损失。其特征为发病急,高热稽留和微血管壁变性、引起全身泛发性小点出血、脾梗死等。世界动物卫生组织(OIE)将本病列入A类法定的传染病,并规定为国际重点检疫对象。在我国制定的《家畜家禽防疫条例实施细则》中也被列为一类传染病。在进行对于猪瘟等致病性和传染性很强的病毒,操作活毒都会面临着高风险,假病毒技术是一种非常有效的研究手段。假病毒是指一种反转录病毒的囊膜糖蛋白被另外一种病毒的囊膜蛋白所置换,而基因组仍保持反转录病毒本身的特性。因为其仅能引起单循环感染(复制缺陷型),作为研究病毒的侵入模型是非常安全的,便于研究病毒的侵入机制、组织嗜性、中和抗体分析以及受体的鉴定等。另外,虽然世界上多采用疫苗免疫来控制猪瘟的流行,但是免疫失败或免疫无效现象常有发生,因此探索新的行之有效的抗病毒策略已经成为当务之急。衣壳蛋白靶向性抗病毒灭活(capsid-targeted antiviral inactivation; CTVI)是近年来兴起的基于胞内免疫的抗病毒策略,其基本原理是在病毒的组装过程中将特定的核酸酶引入到病毒颗粒内部,破坏病毒基因组,从而达到灭活子代病毒的目的。因此本研究构建了猪瘟病毒囊膜糖蛋白的假病毒体系,应用该体系研究了囊膜蛋白对猪瘟病毒侵入细胞的重要性,鉴定了病毒感染的宿主细胞谱；并建立了可替代活病毒在操作上更安全的猪瘟病毒中和试验技术平台,利用该技术平台对临床免疫囊膜糖蛋白E2的B细胞表位亚单位疫苗免疫猪群进行了中和抗体检测。同时,利用CTVI原理,构建了猪瘟病毒衣壳蛋白(Cap)与葡萄球菌核酸酶(SNase)的融合蛋白的PK-15细胞系,通过IFA、Q-PCR和ELISA证明稳定表达融合蛋白的细胞系可以抑制猪瘟病毒的增殖。论文的主要研究内容如下：1.猪瘟病毒囊膜糖蛋白的克隆及其在293T中的表达通过反转录-聚合酶链式反应(RT-PCR)扩增了猪瘟病毒(CSFV) Shimen株囊膜糖蛋白E0、E2和E012三个完整的阅读框基因并进行了克隆与序列鉴定,分别将其插入真核表达载体pcDNA3.0,构建了重组真核表达质粒pcDNA-E0, pcDNA-E2和pcDNA-E012。将此3个质粒经大量提取并纯化后,经磷酸钙转染人胚肾细胞(293T)。采用兔抗猪瘟高免血清为一抗,FITC-SPA为二抗,分别应用流式细胞术(FACS)和免疫转印(Western blot)鉴定真核质粒在293T细胞中的表达。结果表明3个质粒均可在293T细胞中表达,Western blot检测分析到了25.7、41.5和90kDa的3个条带,FACS检测到荧光细胞比例分别为60.2%、55.2%和56.5%。说明囊膜糖蛋白E0、E2和E012均能表达在细胞膜上,为后续假病毒颗粒的形成奠定了基础。2.构建整合囊膜糖蛋白的假型猪瘟病毒体系及其鉴定将上述已经鉴定的重组真核表达质粒pcDNA-E0, pcDNA-E2和pcDNA-E012分别与MuLv假型病毒构建体系的两种骨架载体pHIT60(包括MuLV的结构蛋白基因,即gag和pol)和pHIT111(为MuLV的基因组,还包括一个报告基因LacZ)经磷酸钙瞬时共转染293T细胞,48h后收集假病毒上清,超速离心后纯化假病毒颗粒。用抗CSFV的多抗为一抗,通过Western blot证明了整个囊膜糖蛋白E012能够在假病毒颗粒表面表达,说明E012能够整合到MuLv病毒粒子表面,该假型猪瘟病毒感染SK6、PK-15、ST、BHK21、Vero、COS7、293T和CEF等8种细胞,48h后检测发现只有在猪源细胞SK6、PK-15和ST中标记基因Lac Z能有效表达,表明所构建的假病毒具有感染性,但只能够感染猪源细胞。因此通过该假型猪瘟病毒进一步证实了猪瘟病毒对猪的单一嗜性。另外,纯化后的病毒经Reed-Muench计算其TCID50为104.58。加入各种浓度的NH4Cl(0～30 mmol/L)预先处理PK-15细胞,37℃温育1h,而后加入MuLV-E012作用,48h后,检测Lac Z.表达量；同时设pH依赖性的MuLV-VSV-G为阳性对照。实验结果表明MuLV-E012感染性与NH4Cl浓度存在极显著的线性负相关性,即随着pH值的升高,假型病毒MuLV-E012对PK-15细胞感染能力逐渐降低,而当NH4Cl浓度达到30 mmol/L时MuLV-E012进入宿主细胞几乎被完全抑制。因此通过假型猪瘟病毒证实了猪瘟病毒侵入细胞是受到pH影响的,即是pH值依赖型囊膜病毒。为避免操作活的病毒带来的搞危险性,本研究利用假型猪瘟病毒建立了微量中和试验。标准阴阳性血清和倍比稀释的待检血清56℃灭活30min后,对每份血清进行2倍梯度稀释,分别与假病毒MuLV-E012按1：1混合,4℃过夜。分别取100-tL上述病毒血清混合物一式3份加到96孔板PK15细胞中,建立了微量中和试验,与全病毒微量中和试验进行了比较,实验结果表明所建立的方法能够代替全病毒进行血清中和抗体滴度的测定,能够检测临床血清的猪瘟抗体中和效价。3.假病毒微量中和试验评价CSFV E2 B细胞表位亚单位疫苗免疫效果本研究目的是将E2的B细胞表位a(aa844-865)和b(aa693-716)串联和单独原核表达后研制亚单位疫苗,通过上述建立的假病毒微量中和试验评价亚单位疫苗的免疫效果,鉴定联合表位的优越性。实验优化了含有重组质粒(pET-rE2-a、pET-rE2-b和pET-rE2-ba)的BL21 (DE3)大肠杆菌的表达条件,将重组工程菌(BL21-rE2-a、BL21-rE2-b和BL21-rE2-ba)大量诱导表达,通过His-Bind螯合层析柱纯化融合蛋白,经SDS-PAGE和薄层扫描分析,结果表明：融合蛋白rE2-a, rE2-b和rE2-ba获得了较好的纯化。用猪抗CSFV阳性血清为一抗,HRP-SPA为二抗,DAB显色表明分别在22、22和25 kDa处出现明显条带,与预期大小相符,证实表达纯化的蛋白rE2-a、rE2-b和rE2-ba有良好的抗原性。将此3个重组蛋白分别与SEPPIC 206 VG白油佐剂进行乳化后免疫6周龄猪瘟抗体阴性的三元商品仔猪,间隔2周再疫1次。2免后3周所有实验组用200TCID50的猪瘟石门株进行动物攻毒实验。各组仔猪在初免后间隔7d采血,应用假病毒微量中和试验检测血清中和抗体水平。实验期间,观察攻毒后各组临床症状、发病率、死淘率和保护率,每天测量实验猪的肛温。动物实验结果表明：3个重组蛋白rE2-a (A组)、rE2-b (B组)和rE2-ba (C组)均能诱导仔猪产生中和抗体水平,在攻毒后4周,中和抗体分别达到64、128和256；而疫苗组(D组)为128。重组蛋白组的保护率分别达到80%、100%和100%,D组也达到100%,因此说明B或C组产生的中和抗体达到或超过D组,免疫保护率可以与D组相当。攻毒后,A-C组的仔猪仅出现轻微的发热,但是持续时间不长,几乎全部健活；仅A组1头仔猪在发热过程中因为受到细菌继发感染,其死亡剖检发现有轻微的猪瘟病变,说明A组保护率欠缺。空白对照组(E组)的仔猪没有中和抗体产生,出现明显的猪瘟临床症状,攻毒后3周全部死亡。该结果为猪瘟病毒囊膜糖蛋白E2 B细胞表位亚单位疫苗的临床应用奠定了基础。4.构建稳定表达CSFV Cap蛋白和SNase融合蛋白的细胞系猪瘟病毒为有包膜的RNA病毒,位于病毒颗粒内部的衣壳蛋白与病毒的RNA结合构成病毒的核衣壳,因此我们可以考虑利用CTVI的原理,构建猪瘟病毒衣壳蛋白(cap)与葡萄球菌核酸酶(SNase)的融合蛋白,用于抗猪瘟病毒感染的研究。根据猪瘟病毒核衣壳蛋白(cap)基因序列设计一对引物,RT-PCR扩增获得编码Cap基因的完整阅读框,将其插入到含有金黄色葡萄球菌核酸酶(SNase)基因的真核表达载体pcDNA-SNase中,筛选获得重组质粒pcDNA-Cap-SNase.测序鉴定后,脂质体转染猪肾细胞(PK-15),经终浓度1000μg/mL的G418稳定筛选,建立稳定表达Cap-SNase融合蛋白的细胞系(PK-15/Cap-SNase)。通过RT-PCR、蛋白免疫印迹(Western blot)和间接免疫荧光(IFA)鉴定Cap-SNase融合蛋白的表达,通过体外消化线性阳性质粒DNA对核酸酶活性进行检测。实验结果表明：建立的PK-15/Cap-SNase细胞系可以稳定表达融合蛋白Cap-SNase,该融合蛋白能够被兔抗核衣壳蛋白抗体所识别,并且具有良好的核酸酶活性,能够对线性阳性质粒DNA进行切割。因此,该细胞系的建立为CTVI策略抑制猪瘟病毒的增殖和感染奠定了基础。5.应用衣壳蛋白靶向灭活策略抗猪瘟病毒感染研究将猪瘟病毒Shimen株感染PK-15/Cap-SNase细胞系后,应用间接免疫荧光(IFA)、荧光定量PCR和ELISA方法鉴定CTVI系统抑制猪瘟强毒的增殖效果。实验结果表明,CTVI能有效抑制病毒的繁殖。IFA鉴定病毒感染5d后PK-15/Cap-SNase细胞中产生的子代病毒滴度与正常PK-15细胞相比较下降了102倍,6d后产生的子代病毒滴度与正常PK-15细胞相比较下降了103倍；real-time PCR分析表明,阴性对照正常PK-15细胞无明显的抑制作用,与而稳定表达融合蛋白Cap-SNase的细胞株在病毒进入的3天出现明显的抑制,接种后6天抑制趋向平稳,在第8天抑制率达到78%,差异显著。应用美国IDEXX CSFV Ag ELISA Kit的检测结果：PK-15对照细胞呈现强阳性,而PK-15/Cap-SNase细胞系的ELISA光吸收度数值明显低,呈现弱阳性。因此本研究表明PK-15/Cap-SNase细胞在不同程度上抑制了猪瘟病毒粒子的增殖。这些结果为进一步将衣壳蛋白靶向病毒灭活策略应用于抵抗猪瘟病毒感染奠定了基础。更多还原

【Abstract】 Classical swine fever(CSF) is a kind of acute, febrile and highly contagious disease that caused by the pathogen Pestivirus suis. CSF can cause great damages to the domestic and international pig breeding industry as it is characterized by high-contagious, high morbidity and mortality and wide range of transmission. The clinical symptoms including acute fever, hyperpyrexia, degeneration in micrangium wall, extensive dot-sized hemorrhage within the skin and infarction in the spleen. Office International Des Epizooties(OIE) has categorized CSF as A class infectious diseases according to related laws, and also specified CSF as one of the international quarantine item. According to the Implementating regultions for quarantine and prevention of livestock and poultry disease ordinance, CSF was classified as the Class A infectious disease.The retroviral envelope protein can be exchanged for envelope proteins from non-related viruses, a process called pseudotyping. Pseudotyped viruses with heterogenic glycoprotein incorporated into retroviral particles were proved to be a safe viral entry model, which only go through a single cycle infection(replication-deficient) and acquired the host range of the parent viruses where the glycoprotein were derived, thus they could facilitate the research on viral entry mechanism, viral tropism, neutralization antibody analysis, and receptor identification. In addition, One of the potential strategies, referred to as capsid-targeted viral inactivation (CTVI), is a conceptually powerful antiviral approach. In this strategy, the viral capsid protein is designed as the carrier of a deleterious enzyme, such as a nuclease, a proteinase, or even a single-chain antibody to bind to a native viral protein. These recombinant proteins are targeted specifically to progeny virions during their assembly to prevent the production of infectious viral particles and the subsequent spread of de novo infection. CTVI has been investigated extensively and shown to be a promising antiviral strategy against several important viruses.Therefore, in this study, the pseudotype system of MuLV particles with CSFV E012 was set up and it can be used to study the entry of CSFV. The results shown SK6、PK15 and ST infected were Lac Z positive, indicating viral entry, and revealed the pseudtype virions of MuLV-E012 were infectious. The pseudotyped particles were used to develop an in vitro micro neutralization assay that was both sensitive and specific for CSFV neutralizing antibody. In addition, serum samples from piglets immunized with E2 subunit vaccine were detected by this micro neutralization assay. To explore the feasibility of using capsid-targeted viral inactivation (CTVI) as an antiviral strategy against CSF infection, a stable cell line was constructed for expressing a fusion protein of CSFV capsid (Cap) and Staphylococcus aureus nuclease (SNase). Then we apply it into the study of anti-CSF infections.The contents of the paper contain five parts as following:1. Cloning and eukaryotic expression of the glycoprotein genes of CSFVTwo basic requirements are essential for successful pseudotyping:(1) the glycoprotein has to be incorporated onto the virions, and (2) the pseudotyped virions have to be infectious. Or the system is not useful. The generation of high titer retro viral stocks for the efficient transduction is an important technical for a pseudotype system.In this study, the glycoprotein E0, E2 and E012 genes of CSFV were amplified by RT-PCR and cloned into pMD-18T vector and sequenced. Then three genes were cloned into eukaryotic expression vector pcDNA3.0, designated pcDNA-E0, pcDNA-E2 and pcDNA-E012 respectively. HEK293T cell were transfected with these three plasmid using calcium phosphate method. The cells were collected cells after 48 h, and incubated for 1 h at 4℃with 1:100 CSFV anti-serum. After three washes with PBS/FCS, the cells were incubated with fluorescein-labelled staphylococcal protein A for 1h at 4℃, subsequently subjected to flow cytometry using a FACSCalibur. Meanwhile, protein expression of recombinant plasmids were also determined by Western blot assay. The results showed that the recombinant plasmids pcDNA-E0, pcDNA-E2 and pcDNA-E012, could be effective expressed in HEK293T cells. It provided a foundation to study the function relationship of the EO and E2 glycoproteins.2. Construction of pseudotype CSFV and study on characteristic and application of pseudotype virusThree plasmids, namely pcDNA-EO/2/012, pHIT60 (including the structural genes of MuLV) and pHIT111 (including the retroviral genome, containing LacZ as a reporter) were co-transfected into HEK293T cells for the production of pseudotyped virions with EO/2/012 glycoproteins of CSFV Shimen strain. The retroviral supernatants were harvested at 48 hours post-transfection, filtered through a 0.45μM filter, and used in western blot and infection assays. Parallel transfections were carried out with supernatants produced in absence of a viral envelope and with the vesicular stomatitis virus (VSV) G proteins, which is known to efficiently pseudotype MuLV. Western-blotting revealed only E012 could be expressed on the virions, indicated the glycoprotein E012 was incorporated onto the retroviral virions. Infection test were performed on SK6、PK15、ST、BHK21、Vero、COS7、HEK293T and CEF cells. The results shown SK6、PK15 and ST infected were Lac Z positive, indicating viral entry, and revealed the pseudtype virions of MuLV-E012 were infectious. The pseudotype system of MuLV particles with CSFV E012 was set up and it could be used to study the entry of CSFV. To assess whether the CSFV pseudotyped virus entry is pH-dependent, PK15 cells were treated with well-characterized lysosomotropic agent NH4Cl. The pH dependency of infection was evaluated by pretreating PK15 cells for 1 h with serum-free 1640 containing ammonium chloride at various concentrations (0-30 mM) at 37℃; this was followed by incubation with supernatants containing the pseudotyped viruses in the presence of ammonium chloride at the consistent concentration as in the pretreating procedure. After 2 h, the supernatants were replaced with 1640 containing 10% FBS. The luciferase activity was determined 48 h later as described above. Treatment with 30 mM NH4C1 caused >90% inhibition of infection by MuLV-E012 or MuLV-VSV G. These data indicated the MuLV-E012’s entry may be pH-dependent. The pseudotyped MuLV-E012 particles were used to develop an in vitro microneutralization assay that was both sensitive and specific for CSFV neutralizing antibody. Neutralization titers measured by this assay were highly parallel with those measured by the assay using live csfv high virulence strain. Because the pseudotype assay does not require handling live CSFV virus, it is a useful tool to determine serum neutralizing titers during natural infection and the preclinical evaluation of candidate vaccines.3. B-cell epitopes of classical swine fever virus glycoprotein E2 expressed in Escherichia coli as subunit vaccine induces protection against CSFVBased on sequence analysis, three B-cell epitopes were chosen to be expressed in prokaryotic express system. One of them was multiple epitopes, the others were mono-epitope. Three recombinant expression plasmids expressing thess epitopes were constructed into pET32a vector, designated pET-rE2-a, pET-rE2-b and pET-rE2-ba. They were transformed into host bacterium BL21(DE3). Single colony was chosen to incubate in LB at 37℃, the protein were induced with 0.1mM IPTG when OD600 was 0.6, the cell grown 3 hours in 37℃. The cell were harvested and lysed with ultrasonication, and then clarified by centrifuge at 12000rpm. The supernatant and pellet were analyzed with SDS-PAGE. In all these cases, most of the proteins were found predominantly in inclusion bodies. The proteins were purified on a His·Bind chelation affinity column. The SDS-PAGE analysis indicated three proteins were purified, and displayed a single band with a molecular weight of 22 kDa,22 kDa and 25 kDa, respectively. Western blotting indicated that purified rE2-a, rE2-b and rE2-ba were recognized by CSFV positive sera specifically and reacted strongly. This suggested that the three linear peptides all possessed immunogenicity.Twenty-five 6-week-old piglets with negative CSFV antibody titers, which were purchased for animal expriments and equally divided into five groups. The pigs were acclimatized for 2 weeks, the body temperatures were measured once daily. Three groups were inoculated with purified rE2-a, rE2-b, and rE2-ba. The fourth group was immunized with a commercial vaccine (HCLV) to serve as a positive control. The fifth group was immunized with PBS as a negative control. All three proteins, rE2-a, rE2-b and rE2-ba, were combined with ISA 206 VG adjuvant in immunization. The pigs were inoculated with 50μg of each peptide for the first and second immunization at an interval of 2 weeks. Pigs immunized were inoculated intranasally (mimicking natural infection) with a lethal dose(200 TCID50) of CSFV on day 21 after the second immunization. Serum samples of immunized pigs were collected by jugular venipuncture every week before and after immunization and every week post-challenge. Each sample was heated to inactivate at 56℃for 30 min, and then was subjected to detection by a safe MuLV-E012 neutralization assay. In vivo, all these epitope-based proteins induced an antibody response and protected pigs against lethal challenge with virulent CSFV strain Shimen. The multiple epitope protein rE2-ba showed better protective effect (similar to that of HCLV vaccine) than that of mono-epitope peptide (rE2-a or rE2-b). The results demonstrated that the reactogenic and immunogenic proteins were produced by the prokaryotic system, and CSFV B-cell linear epitope peptides induced immunoregulation, similar to that of attenuated virus. Therefore, CSFV B-cell linear epitope based peptides expressed in a prokaryotic system can be used as immunogens in pigs, and may be used to develop more effective subunit vaccines. 4. Establishment and identification of a stable cell line by CSFV capsid and Staphylococcus aureus nuclease fusion proteinFor construction of the vector expressing the fusion protein, a pair of specific primers were designed and used to amplify the coding region of the Cap. Then the gene Cap was ligated into the expression plasmid pcDNA-SNase and resulted in pcDNA-Cap-SNase plasmid. The positive recombinant products were confirmed by restriction enzyme digestion and DNA sequencing. Then pcDNA-Cap-SNase was transfected into the PK-15 cells. The cell line was passaged continuously for 15 generations or more under G418 selection, which was named as PK-15/Cap-SNase cells. In order to confirm whether the screened PK-15/Cap-SNase cells expressed stably Cap gene (317 bp) and SNase gene (469 bp), PCR was performed using specific primers to amplify the products from the isolated total RNA; however, the products were not detected in Rnase-treated total RNA (negative control), indicating that fusion gene Cap-SNase was transcribed in the PK-15/Cap-SNase cells. To identify further the expression products of the recombinant plasmid, Western blot was carried out using an anti-Cap antibody. A protein band of 31 KDa (Cap=14 KDa and SNase=17 KDa) was detected in the transfected cell lysis. The expressed fusion protein was detected by indirect immunofluorescent signals in the cytoplasm.An in vitro DNA digestion assay was designed to determine nuclease activity. The activity of 5μL of cell lysate containing Cap-SNase was similar to that of 0.5 pg of a standard preparation of SNase, while the linearized plasmid DNA could not be digested when removing Ca+ by EDTA treatment, indicating that the expressed Cap-SNase retained a good Ca2+-dependent nuclease activity.5. Inhibition of replication of CSFV by capsid-targeted virus inactivationPK-15/Cap-SNase and normal PK-15 cells were infected with the CSFV Shimen strain, and then titers of the progeny virus in cell supernatants were detected. The results showed that after infection for 5 d, virus titer produced by PK-15/Cap-SNase cells was 102 lower compared with that of normal PK-15 cells. After 6 d, a greater antiviral effect was observed in the PK-15/Cap-SNase which produced a virus titer that was 103.5 lower than the control (P<0.01). The results of Real-time PCR showed that genomic copy numbers of CSFV in PK-15 cells and PK-15/Cap-SNase cells both increased with time post-infection. The virus in PK-15 cells reached peak at 6dpi then gradually decreased. However, the viral genomic copy number in PK-15/Cap-SNase cells began to decrease at 3dpi, reaching a stable low level at 6 dpi, and the inhibitory rate was 70.8% at 8 dpi. Compared with the parental PK-15 cells, the CSFV genome replication was significantly inhibited in PK-15/Cap-SNase cells (P<0.01). CSFV was positive in the supernatant of PK-15 cell culture but weakly positive in the supernatant of PK-15/Cap-SNase cell culture, further indicating fusion Cap-SNase protein mediated inhibition of CSFV replication. The ELISA results showed stable expression of Cap-SNase could inhibit the proliferation of CSFV virions in PK-15 cells.更多还原