SARS冠状病毒M蛋白的原核表达及其DNA疫苗构建

为探讨SARS-CoV的M蛋白的免疫学特性以及M蛋白作为SARS-CoV病毒疫苗组分的可行性和必要性.分别用pET-15b和pET-22b在大肠杆菌中表达SARS-CoV的M蛋白,亲和层析纯化后作为抗原应用.同时,将M蛋白的编码基因克隆进分泌型真核表达载体pSecTagB中得到重组质粒pSecM作为DNA疫苗,免疫BALB/c小白鼠、制备SARS-CoV M蛋白的抗血清.并用纯化后的M蛋白建立的SARS-CoV M抗体ELISA检测技术研究所构建的M-DNA疫苗的免疫效果.结果表明:两种重组M蛋白在大肠杆菌中均以可溶性形式得到高效表达,经与华大产的用灭活SARS全病毒制备的SARS-CoV抗体ELISA检测试剂盒比较实验,证明该原核表达的重组M蛋白能与SARS确诊病人血清以及M-DNA免疫鼠血清发生特异性抗原抗体反应.这两种重组M蛋白有可能作为抗原组分用于临床SARS-CoV检测中;所构建的SARS-CoV的M基因核酸疫苗能在小鼠体内产生特异性抗体,提示M蛋白在SARS-CoV疫苗尤其是组分疫苗的研制中应加以考虑,为DNA疫苗的开发提供了依据.     

SARS-CoV S蛋白受体结合区的重组表达及免疫原性分析

为了表达SARS-CoV的S蛋白的受体结合区并对其免疫原性进行分析,用PCR方法扩增S蛋白的受体结合区基因片段,克隆至原核表达质粒pET-F32a＋并在大肠杆菌中表达,应用Western—blot鉴定表达的目的蛋白,而后以该蛋白作为诊断抗原包被酶联卡反来检测20份SARS病人血清和28份健康人血清,结果原核表达的S蛋白能够和所用的SARS病人血清反应。这提示表达的S重组蛋白具有良好的抗原性。将变性纯化的重组蛋白和复性蛋白分别皮下免疫小鼠,第三次免疫一周后收集抗血清,用ELISA测定抗体和同时测定中和抗体活性。用变性的抗原免疫的小鼠血清均无中和活性;而用复性的蛋白免疫的小鼠产生了中和抗体。实验表明,S蛋白受体结合区无线性中和表位,中和抗体的产生是由构象表位诱导的。提示该蛋白有可能应用于亚单位疫苗的研究。     

SARS冠状病毒结构蛋白在血清学诊断中潜在应用价值的比较

为了确定特异的SARS抗体检测抗原,比较了SARS冠状病毒(SARS-CoV)主要结构蛋白与SARS患者血清的反应性。从SARS死亡患者的肺组织提取的总RNA为模板,用RT-PCR技术分别扩增S、N、M和E4种结构蛋白基因,对3种S截短突变体和N、M、E的重组蛋白在大肠杆菌中进行表达。以表达的蛋白为抗原,应用Western blot跟踪检测11例SARS患者血清54份。结果显示：SARS—CoV的重组N蛋白和s蛋白有很强的抗原性,s蛋白的3个区段的抗原性强弱存在差异,S3抗原性强于S1和s2;在患病第1周、2周、3周及3周以上,N蛋白和s3蛋白抗体检出率分别为40％、65。2％、100％、100％和40％、61％、76.2％、100％;提示SARS-CoV重组N蛋白和S3蛋白在SARS的血清学诊断中有一定的应用价值。     

SARS冠状病毒S受体结合区蛋白片段在杆状病毒载体中的表达及其抗原性研究

目的:利用Bac-to-Bac1杆状病毒系统,在sf9昆虫细胞中表达严重急性呼吸综合征(SARS)冠状病毒(SARS-CoV)的S受体结合区蛋白片段,并对其免疫原性进行研究。方法:将S蛋白的受体结合区基因片段定向克隆至转座载体pFast-Bac1,转化大肠杆菌DH10Bac感受态细胞,用抗生素平板筛选重组杆粒。脂质体介导重组杆粒转染sf9昆虫细胞,待细胞形态明显改变后收获细胞和培养上清液。利用SARS病人恢复期抗血清做ELISA和Western印迹,分析重组蛋白的抗原性。结果:ELISA和Western印迹表明,在sf9昆虫细胞中表达的SARS-CoVS受体结合区重组蛋白可与SARS病人恢复期抗血清发生特异反应。结论:获得了在昆虫细胞内表达的SARS-CoVS受体结合区重组蛋白,并证明该蛋白有可能用于SARS感染的抗体检测,为SARS-CoV免疫机制及其疫苗的进一步研究奠定了基础。     

SARS冠状病毒NS融合蛋白的重组表达纯化与免疫学性质分析(英文)

刺突蛋白(S)和核心蛋白(N)是SARS冠状病毒的主要结构蛋白.在病毒细胞受体结合和病毒包装过程起重要作用.重组融合表达这2种蛋白具有较高的诊断学价值.对SARS病毒N蛋白和S蛋白氨基酸序列进行计算机分析,选择含有优势抗原表位的N蛋白1～227位氨基酸片段和S蛋白450～650位氨基酸片段,采用序列重叠延伸策略(sequenceoverlappingextension,SOE)构建编码N1227LinkerS450650新型融合蛋白的基因片段,导入原核表达载体,实现融合蛋白在大肠杆菌的高效表达.利用组氨酸标签亲和层析的方法纯化,获得高纯度的融合蛋白.对该融合蛋白的结构特征模拟分析的结果显示,其免疫化学性质均无显著改变.采用ELISA和Western印迹方法对其识别SARS冠状病毒特异性抗体的能力进行初步鉴定,显示该融合蛋白具有较好的抗原性和特异性,可有效特异性地检测恢复期SARS病人血清中抗SARS冠状病毒结构蛋白的抗体,可以作为SARS冠状病毒感染的辅助诊断手段.     

Construction of a eukaryotic expression plasmid encoding partial S gene fragments of the SARS-CoV and its potential utility as a DNA vaccine

The spike (S) protein, a main surface antigen of the SARS coronavirus (SARS-CoV), is considered to be one of the most important protective antigen candidates for targets for vaccine design against the virus. In this study, a secreted recombinant expression plasmid, pVAX-S1, encoding the partial S protein with a signal peptide, was constructed and used to immunize BALB/c mice through electroporation. It was demonstrated that the eukaryotic expression vector pVAX-S1 was successfully constructed by restriction enzyme and sequence analysis. The expressed protein could be detected specifically by Western blot analysis. The serum IgG level of the vaccine group mice was significantly higher than that of the corresponding control group at day 14 after vaccination (P < 0.05). The vaccine group demonstrated significantly higher S1 protein lymphocyte proliferation index (LPI) than the control groups (P < 0.05). Furthermore, in the experimental group, a decrease in the ratio of CD4(+) to CD8(+) T-lymphocytes and an increase level of IFN-gamma in serum were observed. However, interleukin-4 (IL-4) was not detectable in two groups. These results strongly demonstrated that the pVAX-S1 plasmid could induce humoral and cellular immune responses in mice, and may be a potential candidate for a DNA vaccine against the SARS coronavirus.     

Expression of SARS-coronavirus nucleocapsid protein in Escherichia coli and Lactococcus lactis for serodiagnosis and mucosal vaccination

The nucleocapsid (N) protein of the severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) is an important antigen for the early diagnosis of SARS and the development of vaccines. It was expressed in Escherichia coli as a fusion with human glutathione S-transferase (hGST) and was confirmed by Western blotting analysis. This recombinant N protein (hGST-N) was purified and used to measure the SARS-CoV N-specific antibody in the sera of eight SARS patients by enzyme-linked immunosorbent assay. Specific antibody response to this purified recombinant N protein was 100% positive in the SARS patients sera, while none of the control sera from 30 healthy people gave a positive reaction in the same assay. The SARS-CoV N protein was also expressed in Lactococcus lactis in the cytoplasm or secreted into the medium. The N-producing strain MG1363/pSECN and the purified hGST-N protein were respectively administered to mice, either orally or intranasally. Results indicated that orally delivered MG1363/pSECN induced significant N-specific IgG in the sera. In conclusion, our work provides a novel strategy to produce the SARS-CoV N protein for serodiagnosis and for L. lactis-based mucosal vaccines.     

Antibody responses to individual proteins of SARS coronavirus and their neutralization activities

A novel coronavirus, the severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), was identified as the causative agent of SARS. The profile of specific antibodies to individual proteins of the virus is critical to the development of vaccine and diagnostic tools. In this study, 13 recombinant proteins associated with four structural proteins (S, E, M and N) and five putative uncharacterized proteins (3a, 3b, 6, 7a and 9b) of the SARS-CoV were prepared and used for screening and monitoring their specific IgG antibodies in SARS patient sera by protein microarray. Antibodies to proteins S, 3a, N and 9b were detected in the sera from convalescent-phase SARS patients, whereas those to proteins E, M, 3b, 6 and 7a were undetected. In the detectable specific antibodies, anti-S and anti-N were dominant and could persist in the sera of SARS patients until week 30. Among the rabbit antisera to recombinant proteins S3, N, 3a and 9b, only anti-S3 serum showed significant neutralizing activity to the SARS-CoV infection in Vero E6 cells. The results suggest (1) that anti-S and anti-N antibodies are diagnostic markers and in particular that S3 is immunogenic and therefore is a good candidate as a subunit vaccine antigen; and (2) that, from a virus structure viewpoint, the presence in some human sera of antibodies reacting with two recombinant polypeptides, 3a and 9b, supports the hypothesis that they are synthesized during the virus cycle.     

Antigenicity and receptor-binding ability of recombinant SARS coronavirus spike protein

Severe acute respiratory syndrome (SARS) is an emerging infectious disease associated with a novel coronavirus and causing worldwide outbreaks. SARS coronavirus (SARS-CoV) is an enveloped RNA virus, which contains several structural proteins. Among these proteins, spike (S) protein is responsible for binding to specific cellular receptors and is a major antigenic determinant, which induces neutralizing antibody. In order to analyze the antigenicity and receptor-binding ability of SARS-CoV S protein, we expressed the S protein in Escherichia coli using a pET expression vector. After the isopropyl-beta-D-thiogalactoside induction, S protein was expressed in the soluble form and purified by nickel-affinity chromatography to homogeneity. The amount of S protein recovered was 0.2-0.3mg/100ml bacterial culture. The S protein was recognized by sera from SARS patients by ELISA and Western blot, which indicated that recombinant S protein retained its antigenicity. By biotinylated ELISA and Western blot using biotin-labeled S protein as the probe, we identified 130-kDa and 140-kDa proteins in Vero cells that might be the cellular receptors responsible for SARS-CoV infection. Taken together, these results suggested that recombinant S protein exhibited the antigenicity and receptor-binding ability, and it could be a good candidate for further developing SARS vaccine and anti-SARS therapy.     

Isolation and identification of an scFv antibody against nucleocapsid protein of SARS-CoV

To develop reagents for early diagnosis and therapeutic drugs against SARS-associated coronavirus (SARS-CoV), a large (3 x 10(9)) immunized human antibody library was constructed from peripheral blood mononuclear cells from six SARS convalescent patients. A single chain variable fragment antibody (N18) with high affinity against N protein of SARS-CoV was isolated. Sequence analysis revealed that the VL gene was composed of VL3h (V lambda subgroup) and JL2 regions and the VH gene was composed of VH1-69 (VH1 subgroup), D2-15, D3-22 and JH6 regions. Soluble N18 antibody was expressed in Escherichia coli HB2151, purified by Ni-NTA affinity chromatography and verified by SDS-PAGE and Western blot. The potential application for early diagnosis was evaluated using N protein capture ELISA in which N18 antibody demonstrated high sensitive activity in detecting N protein of SARS-CoV. Finally, the potential usefulness of the N18 antibody in prophylaxis, vaccine design and therapy of SARS is discussed.     

Generation and characterization of DNA vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus

Severe acute respiratory syndrome (SARS) is a serious threat to public health and the economy on a global scale. The SARS coronavirus (SARS-CoV) has been identified as the etiological agent for SARS. Thus, vaccination against SARS-CoV may represent an effective approach to controlling SARS. DNA vaccines are an attractive approach for SARS vaccine development, as they offer many advantages over conventional vaccines, including stability, simplicity, and safety. Our investigators have previously shown that DNA vaccination with antigen linked to calreticulin (CRT) dramatically enhances major histocompatibility complex class I presentation of linked antigen to CD8(+) T cells. In this study, we have employed this CRT-based enhancement strategy to create effective DNA vaccines using SARS-CoV nucleocapsid (N) protein as a target antigen. Vaccination with naked CRT/N DNA generated the most potent N-specific humoral and T-cell-mediated immune responses in vaccinated C57BL/6 mice among all of the DNA constructs tested. Furthermore, mice vaccinated with CRT/N DNA were capable of significantly reducing the titer of challenging vaccinia virus expressing the N protein of the SARS virus. These results show that a DNA vaccine encoding CRT linked to a SARS-CoV antigen is capable of generating strong N-specific humoral and cellular immunity and may potentially be useful for control of infection with SARS-CoV.     

Calreticulin as a hydrophilic chimeric molecular adjuvant enhances IgG responses to the spike protein of severe acute respiratory syndrome coronavirus

Fragment 450-650 of the spike (S) protein (S450-650) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains epitopes capable of being recognized by convalescent sera of SARS patients. Vaccination of mice with recombinant S450-650 (rS450-650) can induce Abs against SARS-CoV, although the titer is relatively low. In the present study, a fusion protein linking a fragment (residues 39-272) of murine calreticulin (CRT) to S450-650 in a prokaryotic expression system was created. Compared with target antigen alone, the recombinant fusion product (rS450-650-CRT) has much improved hydrophilicity and immunogenicity. The S450-650-specific IgG Abs of BALB/c mice subcutaneously immunized with rS450-650-CRT were in substantially higher titer (approximately fivefold more). Furthermore, the fusion protein, but not rS450-650 alone, was able to elicit S450-650-specific IgG responses in T cell deficient nude mice. Given that rCRT/39-272 can drive the maturation of bone-marrow-derived dendritic cells, directly activate macrophages and B cells, and also elicit helper T cell responses in vivo, we propose that fragment 39-272 of CRT is an effective molecular adjuvant capable of enhancing target Ag-specific humoral responses in both a T cell-dependent and independent manner. Fusion protein rS450-650-CRT is a potential candidate vaccine against SARS-CoV infection.     

A strategy for searching antigenic regions in the SARS-CoV spike protein

In the face of the worldwide threat of severe acute respiratory syndrome (SARS) to human life, some of the most urgent challenges are to develop fast and accurate analytical methods for early diagnosis of this disease as well as to create a safe anti-viral vaccine for prevention. To these ends, we investigated the antigenicity of the spike protein (S protein), a major structural protein in the SARS-coronavirus (SARS-CoV). Based upon the theoretical analysis for hydrophobicity of the S protein, 18 peptides were synthesized. Using Enzyme-Linked Immunosorbent Assay (ELISA),these peptides were screened in the sera from SARS patients. According to these results, two fragments of the S gene were amplified by PCR and cloned into pET-32a. Both S fragments were expressed in the BL-21 strain and further purified with an affinity chromatography. These recombinant S fragments were confirmed to have positive cross-reactions with SARS sera, either by Western blot or by ELISA. Our results demonstrated that the potenti     

重组SARS冠状病毒M蛋白的表达、纯化及鉴定

SARS冠状病毒是人的严重急性呼吸综合征的病原体。根据对其他种类冠状病毒的研究结果 ,膜蛋白 (M蛋白 )是病毒主要的结构蛋白 ,重组M蛋白可被用来作为抗原检测对应冠状病毒的感染和制备疫苗。SARS病毒M蛋白基因克隆到原核表达载体pMAL cRI中 ,利用N端和C端分别融合麦芽糖结合蛋白 (maltosebindingprotein和MxeGyrAinteinCBD的策略 ,在大肠杆菌中初步表达了重组M蛋白 ,并通过Western印迹和质谱对蛋白质进行了鉴定。重组蛋白质经亲和层析得到了部分纯化 ,纯化后的蛋白质将用于功能研究与诊断试剂盒的研制。     

日本血吸虫新抗原基因的筛选、克隆、 表达和免疫效果研究

为了寻找日本血吸虫 (Schistosoma japonicum, Sj) 新的疫苗候选基因并进行免疫效果研究,用 Sj 雌虫抗原免疫家兔制备血清,对Sj成虫 cDNA 文库进行免疫筛选,将获得的新基因 ( 命名为Sj-F1, GenBank 登录号为 AY261995) 克隆入原核表达载体 pTWIN1 和真核表达载体 pcDNA3 ,经 PCR 、限制性酶切筛选和鉴定阳性重组子. 将 pTWIN1/Sj-F1 质粒转化大肠杆菌 ER2566,在低温和低 IPTG 浓度下诱导表达可溶性重组融合蛋白 (rSj-F1/intein2),并经 SDS- 聚丙烯酰胺凝胶电泳 (SDS-PAGE) 和蛋白质印迹 (Western blot) 分析鉴定. 将 pcDNA3/Sj-F1 质粒转化大肠杆菌 ER2502 ,大量制备 DNA 疫苗. 用重组融合蛋白和 DNA 疫苗免疫小鼠,末次免疫后 2 周用Sj尾蚴进行攻击感染. 感染后 42 天剖杀冲虫,计算减虫率和减卵率. 感染前采血用 ELISA 法检测抗体. 免疫保护效果测定显示：重组蛋白疫苗以 FCA 作佐剂经皮下免疫和以壳聚糖作佐剂经粘膜免疫分别获得了 28.07%、 24.69% 的减虫率和 48.30% 、 46.38% 的减卵率; DNA 疫苗 (pcDNA3/Sj-F1) 单独免疫获得了 18.47% 的减虫率和 35.06% 的减卵率;用 DNA 疫苗启动免疫后用重组蛋白疫苗经皮下加强免疫,减虫率和减卵率分别提高到了 40.42% 和 56.17%;用 DNA 疫苗启动免疫后用重组蛋白疫苗经黏膜加强免疫,减虫率和减卵率增高更明显,分别提高到了 42.38% 和 62.87%. 结果表明,Sj-F1 重组蛋白疫苗及 DNA 疫苗均可诱导小鼠产生部分抗血吸虫感染的保护力,两者联合免疫保护效果优于单一疫苗.     

SARS-CoV核衣壳蛋白的表达与免疫研究

依据GenBank中SARS基因组序列,采用人工合成的方法合成编码SARS病毒N蛋白的全基因(1296bp)序 列,再与设计的CTL特异性表位基因(195bp)重组后,克隆到pET-28a( )质粒中,重组质粒转化到大肠杆菌BL21 (DE3)中诱导表达。利用SARS患者恢复期阳性血清,鉴定表达蛋白。进一步纯化后免疫马,并用ELISA方法检 测血清抗体效价。结果显示SDS-PAGE表明所表达的蛋白相对分子质量约为55000 Da,与预计大小相符;Western blot显示表达的蛋白具有良好的抗原性和特异性。对表达蛋白形成的包涵体进行洗涤,得到的蛋白纯度可达到 70．1％。切胶纯化免疫马后,获得的抗SARS抗体滴度达1:2560。为亚单位疫苗研制和精制抗SARS抗体免疫制 剂提供基础。     

SARS病毒核蛋白基因的克隆及其表达研究

从一例输入性传染性非典型性肺炎病人血清中提取病毒RNA,通过RT—PCR方法扩增出SARS病毒核蛋白基因片段,克隆入质粒载体pUCm—T后,进行核苷酸序列的测定及分析,与已公布的SARS病毒基因序列进行比较,证实为SARS冠状病毒核蛋白基因。为了解该病毒核蛋白的抗原特性,将核蛋白基因插入表达载体,构建重组质粒pET28a—SN,转导大肠杆菌BL21(DE3)后,加IPTG诱导表达。产物经SDS—PAGE电泳分析,表达出相对分子量约为50kDa的蛋白,占整个菌体的45％左右。Westem—blot分析表明,表达产物仅与SARS阳性病人血清起反应,而与正常血清不起反应。间接ELISA免疫检测,抗原滴度达1：12500。表明表达的核蛋白为SARS特异性抗原,这为SARS病毒的诊断试剂的研制提供了方便而安全的抗原来源。     

SARS冠状病毒N蛋白基因的克隆、表达及活性测定

利用PCR基因扩增法,以SARS冠状病毒全基因质粒为模板,获得N蛋白相应抗原基因,构建了表达载体pBV220／SARS-N,并在E．coli中获得高效表达。用纯化后的N蛋白抗原包被测定板,通过间接ELISA法对阴阳性血清进行活性测定,结果表明,在46份阳性血中有41份被测出,检出率为89．13％。本研究克隆并表达了SARS冠状病毒N蛋白,为进一步研制SARS病人抗体检测试剂和SARS疫苗奠定了基础。     

Recombinant tegumental protein Shistosoma japonicum very lowdensity lipoprotein binding protein as a vaccine candidate against Schistosoma japonicum

A polyhistidine-tagged recombinant tegumental protein Schistosoma japonicum very lowdensity lipoprotein binding protein (SVLBP) from adult Schistosoma japonicum was expressed in Escherichia coli. The affinity purified rSVLBP was used to vaccinate mice. The worm numbers and egg deposition recovered from the livers and veins of the immunized mice were 33.5% and 47.6% less than that from control mice, respectively (p<0.05). There was also a marked increase in the antibody response in vaccinated mice: the titer of IgG1 and IgG2a, IgG2b in the vaccinated group was significantly higher than that in the controls (>1:6,400 in total IgG). In a comparison of the reactivity of sera from healthy individuals and patients with rSVLBP, recognition patterns against this parasite tegumental antigen varied among different groups of the individuals. Notably, the average titres of anti-rSVLBP antibody in sera from faecal egg-negative individuals was significantly higher than that in sera from the faecal egg-positives, which may be reflect SVLBP-specific protection. These results suggested that the parasite tegumental protein SVLBP was a promising candidate for further investigation as a vaccine antigen for use against Asian schistosomiasis.     

SARS-CoV单克隆抗体的制备及抗原表位的初步鉴定

参照已发表的SARS冠状病毒BJ01株基因序列 ,利用计算机软件预测并选取该病毒S、M、N三种主要结构蛋白部分抗原性优势区域 ,以编码Gly-Pro-Gly序列相连接合成两段嵌合基因A和B。并分别克隆于pGEX -6p- 1载体上用IPTG进行诱导表达 ,以纯化的嵌合蛋白A和B为抗原 ,分别免疫BALB c小鼠制备单克隆抗体。利用单克隆抗体亚型检测试剂盒和SARS CoV商品化ELISA检测试剂盒对其进行亚型和特异性鉴定。结果表明融合表达两段嵌合基因产物 ,其大小分别为 34kD和35kD ,Westernblot分析证实两种表达产物都能被SARS病人康复期血清所识别。获得了 6株能稳定分泌特异性抗体的阳性细胞克隆株。亚型鉴定结果除D3C5为IgG2a外其他单抗均为IgG1,而且所有单抗的轻链均为κ链。特异性鉴定发现除D3D1外 ,其余的 5株单抗均能与SARS CoV商品化ELISA检测试剂盒发生特异性反应。将D3D1与灭活后经超声波裂解的SARS CoV进行Westernblot分析 ,发现它能特异性识别 180kD的蛋白带。分别融合表达了 6个S蛋白的寡肽 (S1- S6 ) ,并对筛选出的单克隆…