一种展示SARS冠状病毒受体结合区的重组枯草杆菌芽孢的制备及免疫原性分析

目的：利用枯草杆菌芽孢呈递技术制备表达SARS冠状病毒S蛋白受体结合区（RBD）的重组芽孢。方法：将枯草杆菌 CotB 基因构建到基因组整合质粒pDG1664中,再将 RBD 基因连接到 CotB 基因的下游,构建成重组质粒pDG1664-CotB-RBD,通过同源重组整合到PY-79枯草杆菌基因组中;利用红霉素抗性筛选重组菌并进行PCR和DNA测序鉴定,Western印迹鉴定重组菌芽孢表面RBD蛋白的表达情况;用表达RBD的重组芽孢以口服方式免疫小鼠,通过ELISA和流式细胞术检测重组芽孢的免疫原性。结果：制备出枯草杆菌基因组整合了RBD抗原基因的重组菌株RS1931,形成的重组芽孢表达相对分子质量约62×103的CotB-RBD融合蛋白;重组芽孢免疫的小鼠血清RBD抗原特异性IgG抗体滴度在末次免疫后2周可达1∶10880,重组芽孢初免后18周的小鼠脾细胞中IFN-γ＋CD4^＋、IL-4＋CD4^＋和IFN-γ＋CD8^＋T细胞比例上调,表明重组芽孢经口服免疫产生良好的体液免疫和细胞免疫应答。结论：针对SARS冠状病毒S蛋白RBD建立了枯草杆菌芽孢呈递技术方法,制备出在枯草杆菌芽孢表面稳定表达外源RBD蛋白的重组株,获得的重组芽孢具有良好的免疫原性,为开发芽孢呈递型SARS疫苗奠定了基础。     

Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion

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Polymorphism of SARS-CoV Genomes

In this work, severe acute respiratory syndrome associated coronavirus (SARS-CoV) genome BJ202 (AY864806) was completely sequenced. The genome was directly accessed from the stool sample of a patient in Beijing. Comparative genomics methods were used to analyze the sequence variations of 116 SARS-CoV genomes (including BJ202) available in the NCBI Gen-Bank. With the genome sequence of GZ02 as the reference, there were 41 polymorphic sites identified in BJ202 and a total of 278 polymorphic sites present in at least two of the 116 genomes. The distribution of the polymorphic sites was biased over the whole genome. Nearly half of the variations (50.4%, 140/278) clustered in the one third of the whole genome at the 3′ end (19.0 kb-29.7 kb). Regions encoding Orf10-11, Orf3/4, E, M and S protein had the highest mutation rates. A total of 15 PCR products (about 6.0 kb of the genome) including 11 fragments containing 12 known polymorphic sites and 4 fragments without identified polymorphic sites were cloned and sequenced. Results showed that 3 unique polymorphic sites of BJ202 (positions 13 804, 15 031 and 20 792) along with 3 other polymorphic sites (26 428, 26 477 and 27 243) all contained 2 kinds of nucleotides. It is interesting to find that position 18379 which has not been identified to be polymorphic in any of the other 115 published SARS-CoV genomes is actually a polymorphic site. The nucleotide composition of this site is A (8) to G (6). Among 116 SARS-CoV genomes, 18 types of deletions and 2 insertions were identified. Most of them were related to a 300 bp region (27 700-28 000) which encodes parts of the putative ORF9 and ORF10-11. A phylogenetic tree illustrating the divergence of whole BJ202 genome from 115 other completely sequenced SARS-CoVs was also constructed. BJ202 was phylogeneticly closer to BJ01 and LLJ-2004.     

SARS病毒基因组的多态性

对SARS病人粪便样本直接测序,得到SRAS—CoV BJ202全基因组序列（AY864806）。应用比较基因组研究方法对GenBank中公布的115株SARS—CoV基因组序列以及BJ202进行分析。以GZ02序列为参照,发现2个以上基因组中同时存在单核苷酸多态（SNP）位点共278个。多态位点在SARS—CoV基因组中呈偏态分布,大约一半突变位点（50．4％,140／278）发生在基因组3’末端1／3区域。编码Orf10-11、Orf3／4、E蛋白、M蛋白和S蛋白区域突变率较高。克隆并测序含有BJ202基因组12个多态位点的11个cDNA以及4个不含已知多态位点的cDNA片段（15个片段总长度为6．0kb）,结果显示：BJ202特有的3个多态位点（13804、1503l和20792）以及另外3个多态位点（26428、26477和27243）均检出两种不同核苷酸;位点18379虽在已公布的115株SARS—CoV基因组中未发现突变,实际上也是多态位点。14个克隆中有8个克隆该位点为A,6个克隆为G。全部116个SARS—CoV基因组中共有18种缺失类型和2种插入类型。大部分缺失发生在编码ORF9和ORF10-11区域（基因组序列27700—28000bp处）。以邻位连接法（Neighbor-Joining）构建了116株SARS—CoV系统发育树,BJ202与BJ01和LLJ-2004等SARS—CoV的亲缘关系较接近。     

人IFN-β启动子基因报告质粒的构建及SARS-CoV3a和7a蛋白对IFN-β诱生作用的初步研究

3a蛋白和7a蛋白是SARS-CoV的非结构蛋白,分别主要由SARS基因组中ORF 3a 和ORF 7a所编码。在体内和体外感染病毒的细胞中均发现了有3a蛋白的表达。首先将pGL3-Control载体进行了改构,除去了SV40启动子基因,获得了pGL3-Enhancer载体,将获得的IFN-β启动子基因连入载体中,构建了带有人IFN-β启动子基因的荧光素酶报告质粒IP-21,并且通过实验证明所构建的质粒在干扰素的诱导剂NDV的作用下能够表达荧光素酶活性,照度计检测荧光素酶活性增强,从而验证了所构建的重组质粒的有效性。为了观察SARS-CoV非结构蛋白3a和7a对干扰素诱生途径的影响,将IP-21瞬转入稳定表达有3a和7a蛋白的CHO细胞,通过荧光素酶的信号强弱对3a和7a的作用进行分析,结果表明SARS-CoV的3a和7a蛋白能够刺激荧光素酶的表达,即在体外的细胞实验中,3a和7a能有效地激活IFN-β的启动子部分。此结果对进一步研究3a和7a的功能以及对SARS-CoV的致病机制的进一步研究有一定意义。     

Insight into the biological impact of COVID-19 and its vaccines on human health

COVID-19 (coronavirus disease-2019) is a contagious illness that has been declared a global epidemic by the World Health Organization (WHO). The coronavirus causes diseases ranging in severity from the common cold to severe respiratory diseases and death. Coronavirus primarily affects blood pressure by attaching to the angiotensin converting enzyme 2 (ACE 2) receptor. This virus has an impact on multiple organ systems, including the central nervous system, immune system, cardiovascular system, peripheral nervous system, gastrointestinal tract, endocrine system, urinary system, skin, and pregnancy. For the prevention of COVID-19, various vaccines such as viral-like particle vaccines, entire inactivated virus vaccines, viral vector vaccines, live attenuated virus vaccines, subunit vaccines, RNA vaccines, and DNA vaccines are now available. Some of the COVID-19 vaccines are reported to cause a variety of adverse effects that range from mild to severe in nature. SARS-CoV-2 replication is controlled by the RNA-Dependent RNA-Polymerase enzyme (RdRp). The availability of FDA-approved anti-RdRp drugs (Ribavirin, Remdesivir, Sofosbuvir, Galidesivir, and Tenofovir) as potent drugs against SARS-CoV-2 that tightly bind to its RdRp may aid in the treatment of patients and reduce the risk of the mysterious new form of COVID-19 viral infection. RdRp inhibitors, such as remdesivir (an anti-Ebola virus experimental drug) and favipiravir (an anti-influenza drug), inhibit RdRp and thus slow the progression of COVID-19 and associated clinical symptoms, as well as significantly shorten recovery time. Molnupiravir, an orally active RdRp inhibitor and noval broad spectrum antiviral agent, is an isopropyl pro-drug of EIDD-1931 for emergency use. Galidesivir's in vitro and in vivo activities are limited to RNA of human public health concern. Top seeds for antiviral treatments with high potential to combat the SARS-CoV-2 strain include guanosine derivatives (IDX-184), setrobuvir, and YAK. The goal of this review is to compile scattered information on available COVID-19 vaccines and other treatments for protecting the human body from their harmful effects and to provide options for making better choices in a timely manner.     

逆转录套式PCR检测粪便样本中的SARS冠状病毒

为了观察SARS冠状病毒在SARS患者粪便中的存在规律,建立了检测SARS冠状病毒RNA的逆转录-聚合酶链反应(RT-PCR)方法,并应用该方法检测了241份SARS患者粪便样本。部分PCR产物应用测序技术进行验证。RT-PCR的灵敏度为10^-10稀释度的病毒原液(原液为10^8TCID50／ml)。241份粪便样本的总体检出率为24．1％(58／241),其中发病后的前10d和20d的检出率均为50．0％。随着发病时间的延长,阳性检出率呈下降趋势。应用RT-PCR从粪便中检测SARS冠状病毒是可行的,在发病50d以后仍有17．0％左右的阳性检出率,提示SARS恢复期患者具有排毒的可能性,给后续的卫生防疫措施提供了一定的参考数据。     

SARS冠状病毒的变异特性分析

通过对目前所发表或公布的CARS相关资料进行分析,结合所做的一些工作,对SARS-CoV的变异特性、影响变异的可能因素以及变异与Ⅰ临床病程发展的联系进行了探讨。结果显示,SARS．CoV在变异发生频率和动态变化上具有RNA病毒的普遍特性,但由于其在人类的传播时间较短,相对于常见的引起慢性感染的RNA病毒,如丙型肝炎病毒(HCV)、人类免疫缺陷病毒(HIV)等,得到鉴定的变异序列数量相对较少。SARS-CoV5基因尤其是S1区变异程度较高,Ⅳ基因较为保守。就目前有限的研究结果分析,没有发现病毒变异与Ⅰ临床病程发展之间有明确的联系。     

Human monoclonal antibodies as candidate therapeutics against emerging viruses and HIV-1

More than 40 monoclonal antibodies (mAbs) have been approved for a number of disease indications with only one of these (Synagis) — for a viral disease, and not for therapy but for prevention. However, in the last decade novel potent mAbs have been discovered and characterized with potential as therapeutics against viruses of major importance for public health and biosecurity including Hendra virus (HeV), Nipah virus (NiV), severe acute respiratory syndrome coronavirus (SARS-CoV), Ebola virus (EBOV), West Nile virus (WNV), influenza virus (IFV) and human immunodeficiency virus type 1 (HIV-1). Here, we review such mAbs with an emphasis on antibodies of human origin, and highlight recent results as well as technologies and mechanisms related to their potential as therapeutics.     

SARS-CoV N蛋白抗原表位的筛选和鉴定

利用噬菌体展示的线性12肽库从马抗SARS-CoV IgG筛选SARS-CoV的抗原表位.经生物淘洗富集的噬菌体克隆被测序.获得两个共有序列DXXDP和TXTLL.它们分别与SARS-CoV N蛋白341-345和392-396位氨基酸序列高度同源.含共有序列的克隆在ELISA竞争抑制试验中与SARS-CoV N蛋白竞争结合马抗SARS-CoVIgG.将两个共有序列肽通过基因重组技术成功展示到大肠杆菌鞭毛,获得重组菌F1和F2.用重组菌F1和F2免疫接种试验Balb/c小鼠产生的血清均能与SARS-CoVN蛋白特异结合.说明DXXDP和TXTLL是SARS-CoVN蛋白的两个连续抗原表位.