昆虫细胞表达的网状内皮组织增殖症病毒囊膜糖蛋白及其免疫性

利用Bac-to-BacBaculovirusExpressionSystems构建了能表达禽网状内皮组织增殖症病毒(REV)囊膜糖蛋白基因(env)的重组杆状病毒。用REV特异性单克隆抗体分别做间接免疫荧光抗体试验(IFA)和免疫转印试验,均可从感染的Sf9昆虫细胞中检出REVenv。重组杆状病毒感染的Sf9细胞裂解后制备成油乳疫苗免疫SPF鸡后,可以诱发维持45d以上的特异性抗REV抗体,并可抵抗REV病毒感染。     

Silencing of HIV-1 Gene Expression by Sirnas in Transduced Cells

The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into cells causes the specific degradation of an mRNA containing the same sequence. To study dsRNA-mediated gene interference targeted to the env gene (NL4-3: 7490-7508) in HIV-1 infected cells, we constructed tandem-type and hairpin-type siRNA expression vectors, which were under the control of two U6 promoters. We also constructed lentiviral-based siRNA expression vectors for further assessment of their antiviral activity in transduced cells. At both the transient plasmid and lentiviral-mediated RNA expression levels, the siRNA encoding the env fragment exhibited sequence-specific suppression of target gene expression and strongly inhibited (≥90%) HIV-1 infection in the cells, as compared to the antisense RNA expression vector. Targeting the HIV-1 env gene with siRNAs encoding the env gene fragment (7490–7508) might be an effective strategy for gene therapy applications in HIV-1/AIDS treatment and management.     

SHIV-XJ02170感染恒河猴后期的传代特点和env基因变异

目的研究SHIV-XJ02170在中国恒河猴感染后期传代过程中病毒和宿主的变化特点,并分析env基因的序列变异。方法将感染中国恒河猴G0401V后期（5年）的SHIV-XJ02170病毒垂直传代2只猴（G0401V→G0402V→0403V）,同时,剔除G0401V猴CD8＋T细胞使潜伏的病毒大量复制后传代1只猴（G0401V→G0404V）,应用流式细胞术、病毒载量测定、序列分析等方法研究该病毒在猴体内长期适应后的病毒和免疫学指标及序列变异特点。结果 G0401V在感染后期仍能稳定传代,且表现出毒力增强的特点。其传代猴G0402V在传代后41 d死亡,外周血CD4＋T淋巴细胞衰竭,仅为43个/mL,符合艾滋病感染猴快速进展型的特征。剔除体内CD8＋T细胞之后的传代猴G0404V的表现类似G0401V,即长期低水平的病毒血症水平。env基因序列分析发现SHIV-XJ02170在G0401V体内长期适应后发生了可遗传的序列变异,并引起糖基化位点的改变。结论 SHIV-XJ02170在猴体长期适应后的传代过程中表现出向强毒株过渡的特征,为进行SHIV-XJ02170感染性克隆的构建奠定了良好的实验基础。     

A 38 nt region and its flanking sequences within gag of Friend murine leukemia virus are crucial for splicing at the correct 5′ and 3′ splice sites

The genome of the Friend murine leukemia virus (Fr‐MLV) contains a 5′ splice site (5′ss) located at 205 nt and a 3′ss located at 5489 nt. In our previous studies, it was shown that if the HindIII–BglII (879–1904 bp) fragment within gag is deleted from the proA8m1 vector, which carries the entire Fr‐MLV sequence, then cryptic splicing of env‐mRNA occurs. Here, attempts were made to identify the genomic segment(s) in this region that is/are essential to correct splicing. First, vectors with a serially truncated HindIII–BglII fragment were constructed. The vector, in which a 38 bp fragment (1612–1649 bp) is deleted or reversed in proA8m1, only produced splice variants. It was found that a 38 nt region within gag contains important elements that positively regulate splicing at the correct splice sites. Further analyses of a series of vectors carrying the 38 bp fragment and its flanking sequences showed that a region (1183–1611 nt) upstream of the 38 nt fragment also contains sequences that positively or negatively influence splicing at the correct splice sites. The SphI–NdeI (5140–5400 bp) fragment just upstream of the 3′ss was deleted from vectors that carried the 38 bp fragment and its flanking sequences, which yielded correctly spliced mRNA; interestingly, these deleted vectors showed cryptic splicing. These findings suggest that the 5140–5400 nt region located just upstream of the 3′ss is required for the splicing function of the 38 nt fragment and its flanking sequences.     

The M-PMV cytoplasmic targeting-retention signal directs nascent Gag polypeptides to a pericentriolar region of the cell

Intracytoplasmic protein targeting in mammalian cells is critical for organelle function as well as virus assembly, but the signals that mediate it are poorly defined. We show here that Mason-Pfizer monkey virus specifically targets Gag precursor proteins to the pericentriolar region of the cytoplasm in a microtubule dependent process through interactions between a short peptide signal, known as the cytoplasmic targeting-retention signal, and the dynein/dynactin motor complex. The Gag molecules are concentrated in pericentriolar microdomains, where they assemble to form immature capsids. Depletion of Gag from this region by cycloheximide treatment, coupled with the presence of ribosomal clusters that are in close vicinity to the assembling capsids, suggests that the dominant N-terminal cytoplasmic targeting-retention signal functions in a cotranslational manner. Transport of the capsids out of the pericentriolar assembly site requires the env -gene product, and a functional vesicular transport system. A single point mutation that renders the cytoplasmic targeting-retention signal defective abrogates pericentriolar targeting of Gag molecules. Thus the previously defined cytoplasmic targeting-retention signal appears to act as a cotranslational intracellular targeting signal that concentrates Gag proteins at the centriole for assembly of capsids.     

中国HTLV-Ⅰ env基因的克隆及Ⅰ＋Ⅱ型嵌合基因的原核表达与抗原活性检测

为尽快研制出国产HTLV抗体诊断试剂,首先从福建HTLV流行区1名HTLV感染者外周血细胞中克隆出HTLV-Ⅰ的全长膜基因（env）,继而结合文献报道、PSA1软件的新疏水性分析和EPI软件的B细胞表位分析数据,选择了gp46中段开始延伸至gp21N端212个氨基酸（aa185-aa396)的基因,并在3‘端通过（GlySer)2与人工合成的HTLV-Ⅱ型的型特异性表位区基因嵌合,插入原核表达载体pRSET,在E.coli中得到了高效表达目的蛋白产量约占菌体总蛋白的30％。通过Triton-X100洗涤,低湿度尿素逐步变性处理,8mol/L尿素溶解后纯度在75％左右,经电泳洗脱纯化,最终纯度可达95%短期,纯蛋白得率约为40％。经Western blottiong检测,该蛋白对4份HTLV-Ⅰ型和2份HTLV-Ⅱ型均有较强反应,而对4份阴性,血清无反应,从而可能用于研究HTLV抗体诊断试剂盒。     

A亚群禽白血病病毒囊膜基因gp85片段在大肠杆菌中的表达

将RAV－1囊膜基因gp85片段来克隆到表达质粒pET-21d( )中得到重组表达质粒pET-21d-RAV-1env(BelⅡ/SalⅠ）序列分析表明该插入片段的核苷酸序列和阅读框都与RAV－1囊膜基因相应序列相同。用其转化大肠杆菌BL21（DE）3并经IPTG诱导,SDS－PAGE分析表明RAV－1囊膜基因融合蛋白表达产物约20kD,与理论值相符;IPTG诱导起始时间比诱持续时间对表达量的影响更大。     

Human leukemia antigen-A~*0201-restricted epitopes of human endogenous retrovirus W family envelope(HERV-W env) induce strong cytotoxic T lymphocyte responses

Human endogenous retrovirus W family(HERV-W) envelope(env) has been reported to be related to several human diseases, including autoimmune disorders, and it could activate innate immunity.However, there are no reports investigating whether human leukemia antigen(HLA)-A~*0201~+restriction is involved in the immune response caused by HERV-W env in neuropsychiatric diseases. In the present study, HERV-W env-derived epitopes presented by HLA-A~*0201 are described with the potential for use in adoptive immunotherapy. Five peptides displaying HLAA~*0201-binding motifs were predicted using SYFEPITHI and BIMAS, and synthesized. A CCK-8 assay showed peptides W, Q and T promoted lymphocyte proliferation. Stimulation of peripheral blood mononuclear cells from HLA-A~*0201~+ donors with each of these peptides induced peptidespecific CD8~+ T cells. High numbers of IFN-γ-secreting T cells were also detectable after several weekly stimulations with W, Q and T. Besides lysis of HERV-W env-loaded target cells, specific apoptosis was also observed. These data demonstrate that human T cells can be sensitized toward HERV-W env peptides(W, Q and T) and, moreover, pose a high killing potential toward HERV-W env-expressing U251 cells. In conclusion, peptides W Q and T, which are HERV-W env antigenic epitopes, have both antigenicity and immunogenicity, and can cause strong T cell immune responses. Our data strengthen the view that HERV-W env should be considered as an autoantigen that can induce autoimmunity in neuropsychiatric diseases, such as multiple sclerosis and schizophrenia. These data might provide an experimental foundation for a HERV-W env peptide vaccine and new insight into the treatment of neuropsychiatric diseases.     

HIV-1膜蛋白在甲醇型酵母中的表达和抗原性的研究

通过计算机辅助分析了中国广西B/C重组亚型HIV-1病毒株的Env蛋白(共851氨基酸残基)氨基酸的疏水性、潜在的抗原表位,与其它亚型的Env蛋白在氨基酸组成的保守性方面进行了比较,选择了env基因的469-511aa,538-674aa和700-734aa三段保守性及抗原性都较强的氨基酸序列构建成嵌合基因,将嵌合基因构建到毕赤酵母Pichia pastoris表达载体pPICZαB中,利用甲醇诱导表达,对表达的蛋白进行了Wester blot和SDS-PAGE分析,结果表明,三段基因能在毕赤酵母Pichia pastoris中表达,产物为40kD的特异性诱导糖蛋白,通过Ni-sepharose 4B金属Ni螯合层析柱分离纯化表达蛋白,酶联免疫检测结果表明,纯化的抗原有很强的抗原特异性,可以用于HIV检测试剂的研制和开发。