2008年23卷4期
本研究中，基于HIV-1膜抗原（Env）的结构及中和抗体表位，通过点突变及高变区删除的方式制备了11种Env突变体。通过单周期感染中和试验与ELISPOT对原始及突变后Env的免疫原性进行分析，其中5种突变体（dWt，M2，M5-2，M5-1和dM7）诱导针对两种假病毒的中和抗体高于原始株，但只有两种突变体（dWt和M5-2）的增高是有统计学意义的。两种突变体（M2和dM2）诱导的Env特异的T细胞反应高于原始株，但只有M2的的提高是有统计学意义的。由上可知，通过改造可有效的提高Env的细胞及体液免疫原性，为HIV-1疫苗设计提供参考。
Eleven env mutants were designed and generated by site-directed mutagenesis of the regions around NAb epitopes and deletions of variable regions in env. The immunogenicities of the generated mutants were evaluated using single-cycle infection neutralization assays with two pseudoviruses and IFN-γ ELISPOT. Overall, five mutants (dWt, M2, M5-2, M5-1 and dM7) induced higher neutralization activities for both pseudoviruses than plasmid Wt, while only two of the mutants (dWt and M5-2) showed significant differences (P<0.05). Two mutants (M2 and dM2) induced more Env-specific T cells than plasmid Wt. Statistically however, significance was only reached for mutant M2. Thus, properly modified HIV-1 Env may have the potential to induce potent cellular and humoral immune responses.
Eleven env mutants were designed and generated by site-directed mutagenesis of the regions around NAb epitopes and deletions of variable regions in env. The immunogenicities of the generated mutants were evaluated using single-cycle infection neutralization assays with two pseudoviruses and IFN-γ ELISPOT. Overall, five mutants (dWt, M2, M5-2, M5-1 and dM7) induced higher neutralization activities for both pseudoviruses than plasmid Wt, while only two of the mutants (dWt and M5-2) showed significant differences (P<0.05). Two mutants (M2 and dM2) induced more Env-specific T cells than plasmid Wt. Statistically however, significance was only reached for mutant M2. Thus, properly modified HIV-1 Env may have the potential to induce potent cellular and humoral immune responses.
逆转录病毒介导的猪瘟E2蛋白四个抗原区的表达 及其兔体免疫活性分析
2008, 23(4): 247 doi: 10.1007/s12250-008-2956-5
收稿日期: 2008-03-21
录用日期: 2008-05-30
猪瘟病毒E2囊膜糖蛋白是CSFV的主要中和性抗原蛋白,携带有能刺激
机体产生保护性免疫的抗原决定簇。因此，本研究通过逆转录病毒表达系统将猪瘟病毒E2基因的四个主要抗原区在PK15细胞中得到了表达，并通过兔体试验对表达的E2蛋白的免疫活性进行了分析。结果显示，表达的蛋白可被猪瘟阳性血清及猪瘟荧光抗体识别，并且可在兔体内产生特异的抗CSFV抗体，并有强的淋巴细胞增殖反应。在攻毒试验中，由重组蛋白及C-株疫苗免疫的兔子均得到了保护。这一系列的结果表明，逆转录病毒介导表达的猪瘟E2蛋白可诱导出很高的特异性抗体，并显示出与C株疫苗相似的保护力，因此具有良好的开发和应用前景
E2 is an envelope glycoprotein of Classical swine fever virus (CSFV) and contains sequential neutralizing epitopes to induce virus-neutralizing antibodies and mount protective immunity in the natural host. In this study, four antigen domains (ABCD) of the E2 gene was cloned from CSFV Shimen strain into the retroviral vector pBABE puro and expressed in eukaryotic cell (PK15) by an retroviral gene expression system, and the activity of recombinant E2 protein to induce immune responses was evaluated in rabbits. The results indicated that recombinant E2 protein can be recognized by fluorescence antibodies of CSFV and CSFV positive serum (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China) using Western blot, indirect immunofluorescence antibody test (IFAT) and ELISA, Furthermore, anti-CSFV specific antibodies and lymphocyte proliferation were elicited and increased by recombinant protein after vaccination. In the challenge test, all of rabbits vaccinated with recombinant protein and Chinese vaccine strain (C-strain) were fully protected from a rabbit spleen virus challenge. These results indicated that a retroviral-based epitope-vaccine carrying the major antigen domains of E2 is able to induce high level of epitope-specific antibodies and exhibits similar protective capability with that induced by the C-strain, and encourages further work towards the development of a vaccine against CSFV infection.
机体产生保护性免疫的抗原决定簇。因此，本研究通过逆转录病毒表达系统将猪瘟病毒E2基因的四个主要抗原区在PK15细胞中得到了表达，并通过兔体试验对表达的E2蛋白的免疫活性进行了分析。结果显示，表达的蛋白可被猪瘟阳性血清及猪瘟荧光抗体识别，并且可在兔体内产生特异的抗CSFV抗体，并有强的淋巴细胞增殖反应。在攻毒试验中，由重组蛋白及C-株疫苗免疫的兔子均得到了保护。这一系列的结果表明，逆转录病毒介导表达的猪瘟E2蛋白可诱导出很高的特异性抗体，并显示出与C株疫苗相似的保护力，因此具有良好的开发和应用前景
E2 is an envelope glycoprotein of Classical swine fever virus (CSFV) and contains sequential neutralizing epitopes to induce virus-neutralizing antibodies and mount protective immunity in the natural host. In this study, four antigen domains (ABCD) of the E2 gene was cloned from CSFV Shimen strain into the retroviral vector pBABE puro and expressed in eukaryotic cell (PK15) by an retroviral gene expression system, and the activity of recombinant E2 protein to induce immune responses was evaluated in rabbits. The results indicated that recombinant E2 protein can be recognized by fluorescence antibodies of CSFV and CSFV positive serum (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China) using Western blot, indirect immunofluorescence antibody test (IFAT) and ELISA, Furthermore, anti-CSFV specific antibodies and lymphocyte proliferation were elicited and increased by recombinant protein after vaccination. In the challenge test, all of rabbits vaccinated with recombinant protein and Chinese vaccine strain (C-strain) were fully protected from a rabbit spleen virus challenge. These results indicated that a retroviral-based epitope-vaccine carrying the major antigen domains of E2 is able to induce high level of epitope-specific antibodies and exhibits similar protective capability with that induced by the C-strain, and encourages further work towards the development of a vaccine against CSFV infection.
在裂殖酵母中通过密码子优化表达HIV-1 Vif蛋白可以介导APOBEC3G蛋白的降解*
2008, 23(4): 255 doi: 10.1007/s12250-008-2957-4
收稿日期: 2008-03-17
录用日期: 2008-05-04
HIV-1的Vif蛋白和宿主细胞的APOBEC3G蛋白间的相互作用是新近发现的的非常有价值的抗HIV-1靶标。由于人类细胞非常复杂，对于研究蛋白间相互作用具有一定的局限性，因此本研究旨在探索裂殖酵母细胞作为研究HIV-1 Vif蛋白和细胞APOBEC3G蛋白相互作用的模型的可行性。通过在裂殖酵母细胞中将HIV-1 Vif、细胞APOBEC3G蛋白分别与GFP蛋白融合表达，利用荧光显微镜观察细胞内绿色荧光的分布判断两种蛋白在酵母细胞中的定位。通过密码子优化技术在酵母细胞中高量表达HIV-1 Vif蛋白。将Vif蛋白和GFP-APOBEC3G蛋白在同一个酵母细胞同时表达，通过观察细胞内绿色荧光的存在情况判断Vif介导APOBEC3G蛋白的降解情况。同时使用蛋白免疫印迹法检测各种实验条件下蛋白的表达水平。研究结果显示，在酵母细胞中，HIV-1 Vif蛋白主要定位于细胞核中，APOBEC3G在细胞浆中靠近细胞核但位置不完全确定的区域聚集呈点状分布。使用密码子优化技术，可以显著提高Vif蛋白在裂殖酵母细胞中的表达量。当在酵母细胞中高量表达Vif蛋白时，Vif蛋白能够介导APOBEC3G蛋白的降解，说明两种蛋白在酵母细胞和人类细胞中具有类似的功能。这些结果表明裂殖酵细胞是研究Vif和APOBEC3G蛋白间相互作用的很好模型。
Interaction between the HIV-1 Vif protein and the cellular host APOBEC3G protein is a promising target for inhibition of HIV-1 replication. Considering that human cells are a very complicated environment for the study of protein interactions, the goal of this study was to check whether fission yeast could be used as a model cell for studying the Vif-APOBEC3G interaction. Vif and APOBEC3G were expressed in fusion with GFP protein in the S. pombe SP223 strain. Subcellular localizations of Vif and APOBEC3G were observed with fluorescent microscopy. Codon optimization was used to over express the Vif protein in S. pombe cells. The degradation of APOBEC3G mediated by Vif was tested through expressing Vif and GFP-APOBEC3G proteins in the same cell. Western Blot analysis was used to measure the corresponding protein levels under different experimental conditions. The results showed that the Vif protein was predominantly localized in the nucleus of S. pombe cells, APOBEC3G was localized in the cytoplasm and concentrated at punctate bodies that were often in close proximity to the nucleus but were not necessarily restricted from other regions in the cytoplasm. Vif protein expression levels were increased significantly by using codon optimization and APOBEC3G was degraded when Vif was over-expressed in the same S. pombe cells. These results indicate that fission yeast is a good model for studying the interaction between the Vif and APOBEC3G proteins.
Interaction between the HIV-1 Vif protein and the cellular host APOBEC3G protein is a promising target for inhibition of HIV-1 replication. Considering that human cells are a very complicated environment for the study of protein interactions, the goal of this study was to check whether fission yeast could be used as a model cell for studying the Vif-APOBEC3G interaction. Vif and APOBEC3G were expressed in fusion with GFP protein in the S. pombe SP223 strain. Subcellular localizations of Vif and APOBEC3G were observed with fluorescent microscopy. Codon optimization was used to over express the Vif protein in S. pombe cells. The degradation of APOBEC3G mediated by Vif was tested through expressing Vif and GFP-APOBEC3G proteins in the same cell. Western Blot analysis was used to measure the corresponding protein levels under different experimental conditions. The results showed that the Vif protein was predominantly localized in the nucleus of S. pombe cells, APOBEC3G was localized in the cytoplasm and concentrated at punctate bodies that were often in close proximity to the nucleus but were not necessarily restricted from other regions in the cytoplasm. Vif protein expression levels were increased significantly by using codon optimization and APOBEC3G was degraded when Vif was over-expressed in the same S. pombe cells. These results indicate that fission yeast is a good model for studying the interaction between the Vif and APOBEC3G proteins.
为研究我国鸭群中鸭圆环病毒(Duck circovirus，DuCV)的感染情况，根据已发表的序列设计了1对检测引物，对来自福建12个养鸭场因发生鸭传染性浆膜炎而病死的43只番鸭的肝组织进行了PCR检测；同时设计了4对引物对其中的检测阳性样品进行全基因组PCR扩增，获得与预期大小相符的4段DNA片段，经克隆测序拼接后获得DuCV全长基因组序列。PCR检测结果显示43只病死鸭中的34只、12个鸭群中的10个鸭群为DuCV阳性,表明我国鸭群中DuCV感染普遍存在。基因组序列分析表明，DuCV福建株(FJ0601)全长1988bp，具有圆环病毒共同的与病毒复制相关的茎环结构和Rep蛋白保守基序等特征，它与4株中国台湾分离株DuCV（TC1/2002，TC2/2002，TC3/2002和TC4/2002）序列在全基因组水平有97.3%～97.5%的同源性，而与美国株（33753-52）和德国株的同源性仅为82.9%和82.3%。应用Clustal W方法作进化树分析显示，FJ0601株DuCV与4株中国台湾分离株在同一分支，而与德国株和美国株距离较远，位于不同的分支上
HSV-1即刻早期蛋白ICP0、ICP22、ICP27在神经母细胞瘤细胞中的生物学分析
2008, 23(4): 272 doi: 10.1007/s12250-008-2937-8
收稿日期: 2008-01-29
录用日期: 2008-06-10
对ICP0、ICP22和ICP27在神经瘤细胞（SH-SY5Y株）中的表达、定位及其可能相互功能关系进行了初步的观察，并对基于这些观察而得出的结果进行了相关讨论。用脂质体法将pEGFP-ICP0、pEGFP-ICP22和pEGFPICP-27重组质粒和空载体(pEGFP—N2)瞬时转染人神经母细胞瘤细胞株SH-SY5Y，分别于16h、40h、66h、90h和186h在荧光显微镜下观察。重组质粒瞬时转染人神经母细胞瘤细胞，观察到ICP0、ICP22和ICP27在细胞中表达和定位。ICPs 在神经母细胞瘤细胞中的核内定位类似在其它细胞中所表现的特性，即与PML-NB结构相关联。由此可以推论，神经细胞内的转录调控系统是影响病毒感染形式的主要因素。
The three immediate-early proteins of HSV-1, ICP0, ICP22, and ICP27, have specific and pivotal functions in transcriptional activation and inhibition, multiple regulatory and control processes of viral genes. In this paper, the expression and localization of these three proteins were studied in neuroblastoma cells using biochemical assays, and their possible and potential interactive functions are discussed. The data show that the three proteins are localized in different structures, specifically in the PML-NB-associated structure, which is a specific nuclear structure composed of many protein molecules and bound tightly to the nuclear matrix in neuroblastoma cells. The results suggest that the activating and suppressive functions of ICPs are mostly dependent on their transcriptional and regulatory roles, including the PML-NB-associated structure.
The three immediate-early proteins of HSV-1, ICP0, ICP22, and ICP27, have specific and pivotal functions in transcriptional activation and inhibition, multiple regulatory and control processes of viral genes. In this paper, the expression and localization of these three proteins were studied in neuroblastoma cells using biochemical assays, and their possible and potential interactive functions are discussed. The data show that the three proteins are localized in different structures, specifically in the PML-NB-associated structure, which is a specific nuclear structure composed of many protein molecules and bound tightly to the nuclear matrix in neuroblastoma cells. The results suggest that the activating and suppressive functions of ICPs are mostly dependent on their transcriptional and regulatory roles, including the PML-NB-associated structure.
副粘病毒Tianjin株结构蛋白的生物信息学分析
2008, 23(4): 279 doi: 10.1007/s12250-008-2947-6
收稿日期: 2008-02-19
录用日期: 2008-06-04
本研究对副粘病毒Tianjin株NP, P, M, F, HN 和 L六个结构蛋白的氨基酸序列进行了生物信息学分析。Tianjin株与来自于副粘病毒科7个属以及尚未分类的25株病毒各蛋白相应序列的系统进化分析显示：Tianjin株与Respirovirus属的SeV、hPIV-1、hPIV-3和bPIV-3位于同一个分支，且与仙台病毒亲缘关系较近。进一步与14株已知仙台病毒相应序列进行系统进化分析，结果提示Tianjin株不属于现有的三个进化分支，而是独立形为一个新的分支。另外，与已知仙台病毒的六个结构蛋白氨基酸序列相似性比较显示：Tianjin株P蛋白保守性较差，相似性仅为78.7%~91.9%；L蛋白较保守，相似性达96.0%~98.0%。序列比对显示：NP蛋白氨基酸序列中存在15个独特的变异位点，P蛋白存在29个，M蛋白存在6个，F蛋白存在13个，HN蛋白存在18个，L蛋白存在29个。这些结果说明：Tianjin株很可能是仙台病毒新的基因型，但是其结构蛋白氨基酸序列中存在较大的变异，提示Tianjin株可能在生物活性、致病性、免疫性乃至流行病学方面与已知仙台病毒具有较大的差异。
Abstract: The amino acid sequences of the NP, P, M, F, HN and L proteins of the paramyxovirus Tianjin strain were analyzed by using the bioinformatics methods. Phylogenetic analysis based on 6 structural proteins among the Tianjin strain and 25 paramyxoviruses showed that the Tianjin strain belonged to the genus Respirovirus, in the subfamily Paramyxovirinae, and was most closely related to Sendai virus (SeV). Phylogenetic analysis with 14 known SeVs showed that Tianjin strain represented a new evolutionary lineage. Similarities comparisons indicated that Tianjin strain P protein was poorly conserved, sharing only 78.7%~91.9% amino acid identity with the known SeVs, while the L protein was the most conserved, having 96.0%~98.0% amino acid identity with the known SeVs. Alignments of amino acid sequences of 6 structural proteins clearly showed that Tianjin strain possessed many unique amino acid substitutions in their protein sequences, 15 in NP, 29 in P, 6 in M, 13 in F, 18 in HN, and 29 in L. These results revealed that Tianjin strain was most likely a new genotype of SeV. The presence of unique amino acid substitutions suggests that Tianjin strain maybe has a significant difference in biological, pathological, immunological, or epidemiological characteristics from the known SeVs
副粘病毒Tianjin株重组血凝素-神经氨酸酶(HN)的血凝活性和抗原性的研究*
2008, 23(4): 287 doi: 10.1007/s12250-008-2965-4
收稿日期: 2008-03-31
录用日期: 2008-04-26
副粘病毒Tianjin株是仙台病毒的一个新基因型，是1999年从群体性爆发下呼吸道感染而致死的普通棉耳狨猴肺组织中分离到的。急性呼吸道感染的儿童该病毒的IgM阳性率为19.28%，提示此病原体与人类有密切关系。血凝素-神经氨酸酶(HN)是仙台病毒主要的跨膜糖蛋白，与病毒的粘附、穿入和释放有关。为了明确副粘病毒Tianjin株HN的结构与功能的关系，本研究表达了HN膜外区的三个片段HN1、HN2和HN3，对其抗原性、红细胞凝集活性和与甲、乙型流感病毒的交叉反应性进行了初步分析。结果表明重组蛋白HN1、HN2和HN3具有天然抗原性。与HN3和HN1段比较，HN2具有更多的位于天然HN蛋白表面的线性表位。HN3的血凝活性强于HN1和HN2段，而且这种活性不完全依赖于HN3的空间构象。HN1、HN2和HN3与甲、乙型流感病毒标准血清均存在交叉反应性，表明重组蛋白HN1、HN2和HN3不适于用做血清学诊断的特异性抗原。
Paramyxovirus Tianjin strain, a new genotype of Sendai virus, was isolated from the lungs of common cotton-eared marmoset that died of severe respiratory infection in the marmoset colonies. The 19.28% IgM positive rate in the young children with acute respiratory tract infection suggested a close relationship between Tianjin strain and humans. Hemagglutinin-neuraminidase (HN) is its major transmembrane glycoprotein responsible for viral attachment, penetration and release. To clear the relationship between HN structure and function of paramyxovirus Tianjin strain, rHN1, rHN2 and rHN3 overlapping the ectodomain of HN protein were expressed. Their antigenicity and hemaglutination activity, as well as cross reactivity to standard antisera against influenza virus type A, type B were analyzed. The results indicated expressed rHNs have the natural antigenicity. The segment rHN2 possesses more linear epitopes exposed on the surface of the native HN protein than found in segments rHN3 and rHN1. The hemagglutination activity of segment rHN3 is higher than that of segments rHN2 and rHN1, and partially dependent on the three-dimensional conformation of HN3 protein. Cross-reactivity between rHNs and standard antisera against influenza virus type A, type B suggested that rHNs might not be the best alternative as specific antigens to detect virus in clinical serum specimens.
Paramyxovirus Tianjin strain, a new genotype of Sendai virus, was isolated from the lungs of common cotton-eared marmoset that died of severe respiratory infection in the marmoset colonies. The 19.28% IgM positive rate in the young children with acute respiratory tract infection suggested a close relationship between Tianjin strain and humans. Hemagglutinin-neuraminidase (HN) is its major transmembrane glycoprotein responsible for viral attachment, penetration and release. To clear the relationship between HN structure and function of paramyxovirus Tianjin strain, rHN1, rHN2 and rHN3 overlapping the ectodomain of HN protein were expressed. Their antigenicity and hemaglutination activity, as well as cross reactivity to standard antisera against influenza virus type A, type B were analyzed. The results indicated expressed rHNs have the natural antigenicity. The segment rHN2 possesses more linear epitopes exposed on the surface of the native HN protein than found in segments rHN3 and rHN1. The hemagglutination activity of segment rHN3 is higher than that of segments rHN2 and rHN1, and partially dependent on the three-dimensional conformation of HN3 protein. Cross-reactivity between rHNs and standard antisera against influenza virus type A, type B suggested that rHNs might not be the best alternative as specific antigens to detect virus in clinical serum specimens.
表达HIV-1gag基因的DNA及重组仙台病毒载体疫苗的 免疫原性研究*
2008, 23(4): 295 doi: 10.1007/s12250-008-2946-y
收稿日期: 2008-02-19
录用日期: 2008-06-24
DNA 及重组病毒载体疫苗是非常有前景的艾滋病疫苗，因为它们可诱导细胞免疫反应。有研究表明在未接受治疗的HIV-1感染者中Gag特异性细胞毒性T淋巴细胞反应与低病毒血症相关。本研究的目的是构建表达中国B亚型HIV-1流行株gag基因的DNA及重组仙台病毒载体疫苗，将其用于预防或治疗HIV感染。分别构建质粒DNA疫苗pcDNA3.1(+)-gag 及重组仙台病毒载体疫苗(rSeV-gag)。采用Western blotting 方法证实Gag蛋白在两种疫苗中皆可正确有效地表达。将这两种疫苗以不同的方式免疫Balb/c 小鼠，分别采用细胞内细胞因子染色法和ELISA方法检测免疫小鼠中 HIV-1 Gag 特异性 CTL 反应及抗体反应。发现以DNA疫苗初免/rSeV-gag疫苗加强免疫的联合免疫方案可诱导最强而且持久的Gag特异性CTL.及抗体反应，甚至在免疫后9周仍能维持较高水平。上述结果表明DNA与rSeV-gag疫苗联合免疫可能会是一种有前景的艾滋病疫苗方案。
Combinations of DNA and recombinant-viral-vector based vaccines are promising AIDS vaccine methods because of their potential for inducing cellular immune responses. It was found that Gag-specific cytotoxic lymphocyte (CTL) responses were associated with lowering viremia in an untreated HIV-1 infected cohort. The main objectives of our studies were the construction of DNA and recombinant Sendai virus vector (rSeV) vaccines containing a gag gene from the prevalent Thailand subtype B strain in China and trying to use these vaccines for therapeutic and prophylactic vaccines. The candidate plasmid DNA vaccine pcDNA3.1(+)-gag and recombinant Sendai virus vaccine (rSeV-gag) were constructed separately. It was verified by Western blotting analysis that both DNA and rSeV-gag vaccines expressed the HIV-1 Gag protein correctly and efficiently. Balb/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gag-specific CTL responses and antibody levels were detected by intracellular cytokine staining assay and enzyme-linked immunosorbant assay (ELISA) respectively. Combined vaccines in a DNA prime/rSeV-gag boost vaccination regimen induced the strongest and most long-lasting Gag-specific CTL and antibody responses. It maintained relatively high levels even 9 weeks post immunization. This data indicated that the prime-boost regimen with DNA and rSeV-gag vaccines may offer promising HIV vaccine regimens.
Combinations of DNA and recombinant-viral-vector based vaccines are promising AIDS vaccine methods because of their potential for inducing cellular immune responses. It was found that Gag-specific cytotoxic lymphocyte (CTL) responses were associated with lowering viremia in an untreated HIV-1 infected cohort. The main objectives of our studies were the construction of DNA and recombinant Sendai virus vector (rSeV) vaccines containing a gag gene from the prevalent Thailand subtype B strain in China and trying to use these vaccines for therapeutic and prophylactic vaccines. The candidate plasmid DNA vaccine pcDNA3.1(+)-gag and recombinant Sendai virus vaccine (rSeV-gag) were constructed separately. It was verified by Western blotting analysis that both DNA and rSeV-gag vaccines expressed the HIV-1 Gag protein correctly and efficiently. Balb/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gag-specific CTL responses and antibody levels were detected by intracellular cytokine staining assay and enzyme-linked immunosorbant assay (ELISA) respectively. Combined vaccines in a DNA prime/rSeV-gag boost vaccination regimen induced the strongest and most long-lasting Gag-specific CTL and antibody responses. It maintained relatively high levels even 9 weeks post immunization. This data indicated that the prime-boost regimen with DNA and rSeV-gag vaccines may offer promising HIV vaccine regimens.
39卷第1期 (2024年2月)
ISSN 1674-0769
EISSN 1995-820X
CN 42-1760/Q
主编: 石正丽
影响因子: 5.5*
*源于2022年JCR
GALLERY
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