狂犬病毒HEP-dG株的拯救

目的筛选免疫原性高、致病性低的狂犬病疫苗候选株。方法应用反向遗传技术,在狂犬病毒弱毒株HEP-Flury株全基因组3'-N-P-M-G-L-5'的假基因区域(G与L之间),插入一个G基因,构建携带双G基因的全长感染性克隆,其与辅助质粒共转染BHK-21细胞,盲传十代,用荧光抗体和RT-PCR鉴定病毒。结果成功获得携带双G基因的狂犬病毒HEP-dG株,该病毒在BHK-21细胞中稳定生长,其病毒滴度达到1010FFU/mL。结论HEP-dG株会为研制高效的狂犬病病毒基因工程口服疫苗提供了良好的疫苗候选株。     

乙脑病毒野毒株SA14感染性克隆的构建及病毒拯救

目的构建乙脑病毒野毒株SA14感染性克隆并进行病毒拯救。方法分节段合成乙脑病毒野毒株SA14全长cDNA序列,采用分段连接方式构建SA14全长cDNA质粒,以其为模板体外转录RNA并电转染导入BHK21细胞进行病毒拯救,用噬斑试验和间接免疫荧光法鉴定拯救病毒;绘病毒生长曲线,测定拯救病毒对小鼠的神经毒力。结果酶切鉴定质粒模板成功构建,用免疫荧光法检测到恢复病毒包膜蛋白表达,噬斑实验发现乙脑野毒株噬斑直径大于疫苗株;绘制病毒生长曲线,野毒株和疫苗株均在60h达到高峰。用拯救病毒进行动物感染试验,野毒株对小鼠脑内神经毒力LD50为10-1.07,皮下神经毒力LD50为100.33。结论成功建立乙脑野毒株SA14感染性克隆并拯救病毒,为进一步研究乙脑病毒的致病机制等奠定了基础。     

新型冠状病毒RBD区重组流感病毒的拯救及初步鉴定北大核心CSCD

目的利用流感反向遗传技术,以B型流感病毒B/Yamagata/16/88株作为骨架,表达新型冠状病毒(SARSCoV-2)S蛋白的受体结合域(RBD),构建以B型流感病毒为载体的新型冠状病毒疫苗候选株。方法基因合成新冠病毒参考株S蛋白上的RBD基因片段(318-541aa),对B型流感病毒B/Yamagata/16/88/的NS1片段进行设计改造并插入RBD序列,构建重组质粒NS110-RBD。将NS110-RBD与其余7个骨架株重组质粒共转染293T细胞和MDCK细胞,拯救表达新型冠状病毒RBD区重组流感病毒,对拯救出的毒株进行形态学、分子生物学鉴定及病毒滴度和Western blot检测。结果成功拯救出重组流感病毒株并命名为rIBV-NS110-RBD。PCR鉴定RBD目的基因大小正确,测序表明拯救病毒株的序列正确,在透射电镜下观察到拯救病毒株的病毒粒子具有流感病毒粒子的典型特征。经测定,拯救株rIBV-NS110-RBD在MDCK细胞上的病毒滴度为10^(5.5) TCID_(50)/ml,在鸡胚上的病毒滴度为10^(6.5) EID_(50)/ml,血凝效价最高达2_(5);Western Blot检测到RBD的表达,其分子质量为35ku。结论成功拯救出高表达新型冠状病毒RBD蛋白的高血凝效价的重组流感病毒株,该病毒具有流感病毒粒子的典型特征,为以B型流感病毒为载体作为新型冠状病毒疫苗的研发提供了新思路。     

利用反向遗传技术产生表达外源基因的重组SA11轮状病毒

目的 利用反向遗传技术产生NSP1基因插入和表达外源基因的重组SA11轮状病毒,构建可表达外源基因的轮状病毒载体。方法 本研究将轮状病毒SA11株的NSP1基因片段的223位(5端)至1 388位的核苷酸删除,并直接插入连接了终止-再启动元件(P2A)的增强绿色荧光蛋白(EGFP)基因,采用已建立的“12质粒”轮状病毒反向遗传系统拯救NSP1基因插入和表达EGFP的重组SA11轮状病毒。通过观察细胞病变效应和插入基因(EGFP)的荧光表达初步确定重组病毒的成功拯救,再结合电镜的形态学观察、RT-PCR的测序结果及病毒基因组的检测结果,进一步证明重组轮状病毒的成功拯救和传代扩增。利用TCID₅₀法和qRT-PCR法,测定了rSA11和rSA11/NSP1-EGFP的病毒滴度并绘制了基因组复制动力曲线。结果 基于“12质粒系统”成功拯救了重组轮状病毒rSA11/NSP1-EGFP,证明NSP1片段截短并插入外源基因后病毒仍可正常复制,且具有良好的遗传稳定性,但病毒滴度低于其亲本株。结论 基于NSP1基因插入和表达EGFP的重组轮状病毒能够实现稳定遗传,为利用反向遗传学...     

狂犬病病毒P基因的原核表达及间接ELISA方法的应用

目的以狂犬病病毒P蛋白作为检测抗原,用间接ELISA方法检测狂犬病病毒抗体。方法根据GenBank发表的狂犬病病毒(Rabies Virus,RV)LEP-Flury株的基因序列设计引物,通过RT-PCR扩增出P基因的全长序列,克隆于pGM-T载体中,获得重组质粒pGM-T-P,将重组质粒用限制性内切酶NotI和EcoRI进行双酶切,酶切产物定向克隆于原核表达载体pET-32a(+)中,构建原核重组表达质粒pET-32a-P,阳性重组质粒转化原核表达宿主菌BL21(DE3),用IPTG诱导表达。SDS-PAGE和Western-blot分析确定蛋白表达量和特异性。用纯化的蛋白作为诊断抗原,通过对反应条件的优化,初步将间接ELISA方法应用于狂犬病病毒抗体的检测中。结果扩增RV P基因,构建了克隆质粒pGM-T-P、原核表达质粒pET-32a-P,高效表达了主要以可溶性形式存在的P蛋白,并能与RV阳性血清发生特异性反应。用表达的狂犬病病毒重组P蛋白建立了用于RV抗体检测的间接ELISA方法。结论成功表达了磷蛋白,用其作为固化抗原以间接ELISA方法检测RV抗体。     

表达IL-33蛋白重组狂犬病病毒的构建及鉴定

目的以狂犬病病毒(RABV)弱毒株SAD为载体,构建表达IL-33蛋白的重组狂犬病病毒株SAD-IL33。方法利用反向遗传学技术,通过Pfl23Ⅱ和NheⅠ位点经酶切连接,将鼠源IL-33基因ORF区插入狂犬病病毒SAD株的伪基因区,获得全长感染性克隆,与辅助质粒共转染BHK-21细胞,拯救重组病毒SAD-IL33,采用RT-PCR、免疫荧光及Western blot鉴定SAD-IL33的生物学特性,并用其感染小鼠,每天观察小鼠体重及精神状态的变化,初步评价其安全性。结果 PCR和测序证实,含IL-33基因的感染性克隆SAD-IL33构建成功;经RT-PCR、免疫荧光及Western blot鉴定,重组病毒SAD-IL33可扩增出1 065 bp的预期条带,同时有RABV G蛋白和IL-33蛋白的特异性荧光,且分子质量单位为70 ku和30 ku,与预期大小一致;SAD-IL33的细胞适应性良好,在BHK-21细胞和Neuro-2a细胞上均能生长,滴度峰值分别为10~(8.345)FFU/ml和10~9FFU/ml,均高于亲本SAD株。将其在BHK-21细胞上连续传代10次,第3～10代的病毒滴度介于10~6～10~8 FFU/ml之间。小鼠感染SAD-IL33后,21 d内体重及精神状态无异常变化。结论成功构建表达IL-33蛋白的重组狂犬病毒SAD-IL33,细胞适应性良好且滴度高,传代稳定,对小鼠无明显影响,为进一步研制安全、高效、廉价的狂犬病预防疫苗奠定了基础。     

狂犬病病毒HEP-Flury-mEG株的拯救与鉴定

目的拯救狂犬病毒HEP-Flury-mEG株,为疫苗制备奠定基础。方法将ERA株G蛋白333位毒力位点修饰为谷氨酸后替换至HEP-Flury株基因组。重组感染性克隆质粒和4个辅助质粒,共转染BHK-21细胞,拯救重组病毒。结果 IFA鉴定成功拯救出了HEP-Flury-mEG株狂犬病病毒。重组病毒G基因经XholⅠ酶切,片段大小为1 071bp和520bp,与预期结果相符。重组病毒在BHK-21细胞中传代4次,滴度维持在1×107.5 FFU/ml。重组病毒经常规负染后在电镜下为弹状粒子,长度和直径与亲本株一致。结论获得了重组狂犬病毒HEP-Flury-mEG株。该病毒滴度高,形态完整,传代稳定,为进一步研究狂犬病毒病毒的生物学特性和新型基因工程疫苗奠定了基础。     

肠道病毒71型(EV71)感染性克隆的构建

目的构建肠道病毒71(enterovrius 71,EV71)的感染性克隆,为研究EV71毒力基因及新型疫苗设计等建立一个技术平台。方法用RT-PCR方法扩增出EV71FJ08149株全基因组,通过TA克隆组装进TOPO-XL-PCR载体中,获得全长cDNA克隆pFJ08149T5和pFJ08149T25。应用T7聚合酶系统在体外将线性化后全长cDNA克隆转录出RNA并转染RD细胞。通过间接免疫荧光试验、RT-PCR、序列测定及免疫电镜等对拯救病毒进行鉴定。结果 RNA转染后72h观察到典型的肠道病毒致细胞病变,拯救病毒经RT-PCR、间接免疫荧光和免疫电镜检测鉴定为EV71型,表明已成功构建了EV71感染性克隆。结论本研究成功构建出具有感染性的EV71全长cDNA克隆,为深入研究EV71的致病机制等提供了有利的工具。     

表达IL-33蛋白重组狂犬病病毒的构建及鉴定北大核心CSCD

目的以狂犬病病毒(RABV)弱毒株SAD为载体,构建表达IL-33蛋白的重组狂犬病病毒株SAD-IL33。方法利用反向遗传学技术,通过Pfl23Ⅱ和NheⅠ位点经酶切连接,将鼠源IL-33基因ORF区插入狂犬病病毒SAD株的伪基因区,获得全长感染性克隆,与辅助质粒共转染BHK-21细胞,拯救重组病毒SAD-IL33,采用RT-PCR、免疫荧光及Western blot鉴定SAD-IL33的生物学特性,并用其感染小鼠,每天观察小鼠体重及精神状态的变化,初步评价其安全性。结果PCR和测序证实,含IL-33基因的感染性克隆SAD-IL33构建成功;经RT-PCR、免疫荧光及Western blot鉴定,重组病毒SAD-IL33可扩增出1065 bp的预期条带,同时有RABV G蛋白和IL-33蛋白的特异性荧光,且分子质量单位为70 ku和30 ku,与预期大小一致;SAD-IL33的细胞适应性良好,在BHK-21细胞和Neuro-2a细胞上均能生长,滴度峰值分别为108.345FFU/ml和109FFU/ml,均高于亲本SAD株。将其在BHK-21细胞上连续传代10次,第3~10代的病毒滴度介于106~108 FFU/ml之间。小鼠感染SAD-IL33后,21 d内体重及精神状态无异常变化。结论成功构建表达IL-33蛋白的重组狂犬病毒SAD-IL33,细胞适应性良好且滴度高,传代稳定,对小鼠无明显影响,为进一步研制安全、高效、廉价的狂犬病预防疫苗奠定了基础。     

狂犬病毒载体的构建与鉴定

目的构建狂犬病毒载体,为新型疫苗研究提供依据。方法将狂犬病毒减毒株SAE基因组分段扩增,然后经逐步拼接得到全基因组克隆,并在其中引入转录终止-起始元件和多克隆位点获得重组质粒pSAETOPO。将绿色荧光蛋白(EGFP)基因和西尼罗病毒prME基因分别克隆至pSAETOPO,然后将带有这两个基因的狂犬病毒全长cDNA切下并克隆至pcDNA3.1(+)载体。将获得的重组质粒pcSAE-GFP和pcSAE-ME转染细胞,分别观察辅助病毒对外源基因表达的影响。结果获得狂犬病毒载体质粒pcSAE-GFP和pcSAE-ME,经酶切分析和序列测定表明所构建克隆是正确的。pcSAE-GFP及pcSAE-ME转染细胞后,在辅助病毒存在下可表达相应外源基因。结论构建的狂犬病毒载体质粒可表达外源基因,为进一步获得表达外源基因的重组狂犬病毒奠定了基础。     

Reverse genetics system for introduction of site-specific mutations into the double-stranded RNA genome of infectious rotavirus

We describe here the successful establishment of a reverse genetics system for rotavirus (RV), a member of the Reoviridae family whose genome consists of 10-12 segmented dsRNA. The system is based on the recombinant vaccinia virus T7 RNA polymerase-driven procedure for supplying artificial viral mRNA in the cytoplasm. With the aid of helper virus (human RV strain KU) infection, intracellularly transcribed full-length VP4 mRNA of simian RV strain SA11 resulted in the rescue of the KU-based transfectant virus carrying the SA11 VP4 RNA segment derived from cDNA. In addition to the rescued transfectant virus with the authentic SA11 VP4 gene, three more infectious RV transfectants, into which silent mutation(s) were introduced to destroy both or one of the two restriction enzyme sites as gene markers in the SA11 VP4 genome, were also rescued with this method. The ability to artificially manipulate the RV genome will greatly increase the understanding of the replication and the pathogenicity of RV and will provide a tool for the design of attenuated vaccine vectors.     

Human Rotavirus Reverse Genetics Systems to Study Viral Replication and Pathogenesis

Human rotaviruses (HuRVAs) are highly important causes of acute gastroenteritis in infants and young children worldwide. A lack of reliable and reproducible reverse genetics systems for HuRVAs has limited a proper understanding of HuRVA biology and also the rational design of live-attenuated vaccines. Since the development of the first reverse genetics system for RVAs (partially plasmid-based reverse genetics system) in 2006, there have been many efforts with the goal of generating infectious recombinant HuRVAs entirely from cloned cDNAs. However, the establishment of a HuRVA reverse genetics system was very challenging until 2019. This review article provides an overview of the historical background of the recent development of long-awaited HuRVA reverse genetics systems, beginning with the generation of recombinant human-simian reassortant RVAs with the aid of a helper virus in 2006 and the generation of recombinant animal (simian) RVAs in a helper virus-free manner in 2017, and culminating in the generation of recombinant HuRVAs entirely from plasmid cDNAs in 2019. Notably, the original HuRVA reverse genetics system has already been optimized to increase the efficiency of virus generation. Although the application of HuRVA reverse genetics systems has only just been initiated, these technologies will help to answer HuRVA research questions regarding viral replication and pathogenicity that could not be addressed before, and to develop next-generation vaccines and intestine-specific rotaviral vectors.     

Recombinant rhabdoviruses as potential vaccines for HIV-1 and other diseases

The failure to develop vaccines to protect against important infectious diseases such as human immunodeficiency virus type I (HIV-1) or Hepatitis C virus (HCV) has increased the interest in new vaccine strategies. One of these methods is immunization with an attenuated recombinant viral vector expressing a foreign antigen, which could protect individuals from later exposure to the respective pathogen. A new method to recover a non-segmented negative-stranded RNA virus (NNSV) from cDNA was described for the first time for rabies virus (RV), a member of the rhabdovirus family. The same approach was successfully used for another rhabdovirus, vesicular stomatitis virus (VSV), and opened the possibility to use rhabdoviruses as vaccine vehicles and biomedical tools. Further research showed that the genomes of rhabdoviruses are highly flexible, easy to manipulate, and able to express large and even multiple foreign genes, and therefore are excellent vaccine candidates. In addition, it has been shown for both RV and VSV that their single surface glycoprotein G, which is responsible for attachment and fusion to the host cell, can functionally be replaced by other viral or cellular glycoproteins. This review gives an overview of the use of RV and VSV as promising new candidates in the fight against HIV-1 and other human diseases.