汉滩病毒核衣壳蛋白C-端T细胞表位鉴定

目的　鉴定汉滩病毒核衣壳蛋白 (HTNVNP)C 端T细胞表位 ,为肾综合征出血热(HFRS)发病机理、疫苗研制及抗病毒免疫反应研究奠定基础。方法　采用Ficoll密度梯度离心法分离HFRS恢复期患者外周血单个核细胞 (PBMC)。用IFN γELISPOT实验和T细胞增殖实验 ,测试 7名患者PBMC对 2 3条NPC 端合成多肽的T细胞应答。结果　IFN γELISPOT实验结果表明 ,2名供体(3、4 )可分别检测到对 5 1、70号 2条多肽特异性T细胞应答。在供体 3,70号肽特异性T细胞频率为4 5SFC 10 6 PBMC ;在供体 4 ,5 1号肽特异性T细胞频率为 82SFC 10 6 PBMC。T细胞增殖实验与ELISPOT结果基本一致 ,但 5 3号肽和 6 4号肽还可分别刺激供体 1和供体 4的T细胞增殖 ,而未能诱导IFN γ分泌。结论　 5 1号和 70号多肽可能是NPC 端较强的T细胞表位。     

HFRS患者汉滩病毒核蛋白特异性CTL克隆的建立及其初步研究

为探讨汉滩病毒核衣壳蛋白 (HTNV NP )诱生的特异性细胞免疫的分子基础 ,为阐明HFRS的发病机制和疫苗的设计提供资料 ,本文分离HFRS患者的PBMC ,建立HTNV NP特异性的CTL克隆 ,同时将克隆有HTNVS基因不同片段的真核表达载体转染患者自身的B淋巴母细胞系 (BLCL ) ,建立CTL靶细胞系 ,并进行细胞杀伤试验。建立的特异性CTL克隆对表达完整NP、NP羧基和氨基端肽段的靶细胞均有比较明显的杀伤效应 ,平均杀伤率分别为 5 0 2 %、 39 0 %和 2 5 8%。表明HTNV NP优势T细胞表位可能主要位于病毒核蛋白的羧基端     

HFRS患者汉滩病毒核蛋白特异性T细胞克隆及其靶细胞的建立

目的　建立肾综合征出血热 (HFRS)患者汉滩病毒 (HTNV)核衣壳蛋白 (NP)特异性CTL克隆 ,为HTNVNPT细胞表位鉴定及HFRS患者T细胞免疫功能研究奠定基础。方法　采用Fi coll密度梯度离心法分离HFRS患者外周血单个核细胞 (PBMC) ,用灭活HTNV和IL 2体外刺激 ,有限稀释法建立T细胞克隆 ,流式细胞术鉴定克隆表型。并用EB病毒 (EBV)转化B淋巴细胞 ,建立B淋巴母细胞样细胞系 (B LCL) ,以含HTNVS基因的重组痘苗病毒感染B LCL作靶细胞 ,以CTL克隆作效应细胞进行细胞毒杀伤试验 ,测定T细胞克隆的抗原特异性。结果　T细胞克隆能特异性识别表达NP的B LCL。在 5名患者中 ,3名有较高的杀伤率 ,并自 2名患者建立了 5株HTNV特异性CTL克隆 ,其表型为CD8 均大于 6 0 %。结论　成功建立了HFRS患者HTNVNP特异性CTL克隆及其靶细胞。NP是HFRS患者HTNV特异性CTL应答的主要靶抗原之一。     

汉滩病毒部分核蛋白基因原核表达产物 的免疫及其保护作用

目的 探讨汉滩病毒（HTNV）部分核蛋白（NP）基因（37－294bp)原核表达产物NP AA1－86多肽的免疫原性及其免疫保护作用。方法 大肠杆菌表达的HTNV部分核蛋白多肽NP AA1－86混合福氏佐剂,腹腔注射免疫BALB／c小鼠,IFA检测抗体应答;^3H-TdR掺入及4h ^51Cr释放试验检测免疫小鼠脾细胞对HTNV的特异淋巴细胞增殖转化指数及细胞毒T淋巴细胞（CTL）杀伤功能;免疫小鼠脾细胞转输感染HTNV A9株的乳鼠,观察对乳鼠致死性的免疫保护作用。结果 免疫后1周小鼠即出现抗体应答反应,抗体滴度最高达1：12800,与重组完整NP免疫组差异无显著性（P＞0．05）;免疫小鼠的淋巴细胞增殖转化指数、CTL杀伤率较对照组明显增高（P＜0．01）,与完整NP免疫组差异无显著性（P＞0．05）,CTL杀伤率随效：靶比例的增高呈逐渐上升趋势;免疫小鼠脾细胞转输可部分保护病毒致死性感染的乳鼠,保护率约25％。结论 大肠杆菌表达的汉滩病毒部分核蛋白多肽NP AA1－86不仅包含NP几乎所有的体液免疫表位,同时含有部分细胞免疫表位,对汉滩病毒感染可产生部分免疫保护作用。     

SARS病毒核衣壳蛋白抗体消长规律

研究SARS病毒抗体的消长规律对于评价患者的愈后及SARS疫苗的使用效果具有重要意义。本项目选用了军事医学科学院生物工程研究所研制的双抗原夹心SARSN蛋白抗体ELISA诊断试剂。对2 79例临床确诊的、发病不同时间SARS患者的血清进行检测,并同时跟踪检测了4 1例SARS患者在不同发病时间(发病3d至16 0d)的血清标本,对抗体产生的时间规律进行了研究。材料和方法标本来源:所用的SARS病例标本为本院检验科从2 0 0 3年2月7日开始,从本院收治病人及广州市中山二院、177医院、广州市第八人民医院、北京小汤山医院收集的SARS病人血清标本…     

汉滩病毒S基因真核表达载体的构建及免疫小鼠的初？…

目的 研究汉滩病毒Ｓ片段编码区基因免疫小鼠的作用。方法 利用基因重组技术，构建含汉滩病毒Ｓ片段编码区基因的真核表达质粒。用此质粒直接注射到ＢＡＬＢ／ｃ小鼠骨骼肌内进行ＤＮＡ免疫，用间接免疫荧光法检测免疫小鼠血清中汉滩病毒抗体。结果 在初次免疫的小鼠血清中检测出有低滴度的汉滩病毒抗体。加强免疫后，抗体水平显著上升，免疫荧光抗体（ＩＦＡＴ）滴度最高达１：６４０。约８７％的免疫动物发生了血清抗体阳转。抗     

汉滩病毒结构蛋白昆虫细胞融合表达产物的免疫原性评价

目的：研究汉滩病毒（HTNV）囊膜糖蛋白G1与核蛋白（NP）部分片段在昆虫细胞中融合表达产物的免疫学特性，为HTNV基因工程疫苗的研制提供依据。方法：构建含有HTNV G1S0.7嵌合基因的重组杆状病毒Bac-G1S0.7，用其感染的Sf9细胞免疫Balb/c小鼠。用间接免疫荧光法、ELISA法、微量细胞培养中和试验及T淋巴细胞增殖试验对被免疫小鼠的体液免疫及细胞免疫应答效果进行检测。结果：Bac-G1S0.7感染的昆虫细胞免疫小鼠后，测得免疫鼠血清抗HTNV抗体滴度最高达1：3200，含有特异性抗HTNV NP及抗HTNV糖蛋白G1的抗体，被免疫小鼠中有部分产生了较低滴度的中和抗体，免疫鼠脾细胞对HTNVNP或糖蛋白的增殖反应指数均高于阴性对照组。结论：G1S.07嵌合基因的昆虫细胞表达产物具有较好的免疫原性，可刺激机体同时产生针对HTNV NP与囊膜糖蛋白G1的细胞及体液免疫应答。     

汉滩病毒S基因真核表达载体的构建及免疫小鼠的初步研究

目的研究汉滩病毒Ｓ片段编码区基因免疫小鼠的作用。方法利用基因重组技术,构建含汉滩病毒Ｓ片段编码区基因的真核表达质粒。用此质粒直接注射到ＢＡＬＢ／ｃ小鼠骨骼肌内进行ＤＮＡ免疫,用间接免疫荧光法检测免疫小鼠血清中汉滩病毒抗体。结果在初次免疫的小鼠血清中检测出有低滴度的汉滩病毒抗体。加强免疫后,抗体水平显著上升,免疫荧光抗体（ＩＦＡＴ）滴度最高达１６４０。约８７％的免疫动物发生了血清抗体阳转。抗体水平及小鼠血清抗体阳转率与注射的质粒ＤＮＡ剂量及注射次数有关。结论应用基因免疫技术能够诱导机体产生针对汉滩病毒的特异性免疫应答     

汉滩病毒M、S基因部分片段嵌合基因真核载体的构建与基因免疫

目的 在本室前期工作的基础上,进一步进行汉滩病毒M基因G2片段与S基因0．7Kb片段嵌合基因基因免疫的研究。方法 构建汉滩病毒76－118株M基因G2片段与S基因5‘端700bp片段的嵌合基因真核表达载体pcDNA3.1-G2S0.7。用该质粒免疫Balb/c小鼠,并用ELISA法及淋巴细胞增殖实验,检测基因免疫后的免疫应答效果。结果 限制性内切酶鉴定结果表明真核表达载体的构建正确。用pcDNA3.1-G2S0.7直接免疫小鼠。可诱导产生抗汉滩病毒核蛋白（NP）及糖蛋白（GP）特异性的抗体,抗体效价分别为1：200及1：80。淋巴细胞增殖实验表明,嵌合基因免疫小鼠脾细胞对NP及GP的增殖指数,均明显高于对照组。结论汉滩病毒M基因G2片段及S基因0．7kb片段的嵌合基因,既可刺激机体产生特异性的抗汉滩病毒体液免疫应答,也可刺激机体产生特异性的细胞免疫应答,本研究为进一步进行汉滩病毒基因疫苗的研究奠定了实验基础。     

The induction and characterization of monoclonal antibodies specific to GP of Ebola virus

The Ebola virus is highly infectious and characterized by hemorrhagic fever, headache, and so on with a high mortality rate. Currently, there are neither therapeutic drugs or vaccines against the Ebola virus nor fast diagnostic methods for the detection of Ebola virus infection. This study reported the induction and isolation of two monoclonal antibodies that specifically recognized the glycoprotein (GP) and secreted glycoprotein (sGP) of the Ebola virus. Plasmids encoding either GP or sGP were constructed and immunized BALB/c mice, accordingly purified sGP was boosted. The antisera were analyzed for binding activity against sGP protein in enzyme-linked immunosorbent assay (ELISA) and neutralization activity in a pseudotyped virus neutralization assay. A number of reactive clones were isolated and two monoclonal antibodies T231 and T242 were identified to react with both GP and sGP. Western blot and ELISA assays showed that the monoclonal antibodies could react with GP and sGP, respectively. Moreover, they could recognize Ebola pseudovirus by cellular immunochemistry assay. We labeled the monoclonal antibody T231 with biotin and analyzed the competitiveness of the two antibodies by the ELISA test. The results showed that the binding epitopes of the two monoclonal antibodies to sGP were partially overlapped. In summary, two GP-specific mAbs were identified, which will be used to detect the Ebola virus or investigate GP.     

Enhanced immune responses of mice inoculated recombinant adenoviruses expressing GP5 by fusion with GP3 and/or GP4 of PRRS virus

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important causes of economic losses of the swine industry. PRRS virus (PRRSV) infection poses a challenge to current vaccination strategies. In this study, three replication-defective adenovirus recombinants expressing fusion protein GP3-GP5, GP4-GP5, or GP3-GP4-GP5 were developed as potential vaccine against PRRSV in a mouse model. Six groups of BALB/c mice (24mice per group) were inoculated subcutaneously twice at 2-week intervals with above mentioned recombinants and other adenoviruses expressing single GP3, GP4, or GP5 protein. The results showed that the mice inoculated with recombinant adenoviruses developed PRRSV-specific antibodies, cellular immune response by 2 weeks post-boost-immunization. However, mice immunized with recombinant adenoviruses rAd-GP3-GP5, rAd-GP4-GP5, and rAd-GP3-GP4-GP5 developed significantly higher titers of neutralizing antibodies to PRRSV and produced stronger lymphocyte proliferation responses compared to mice immunized with rAd-GP3, rAd-GP4 or rAd-GP5 alone. It was also found that mice immunized with rAd-GP3-GP5 and rAd-GP3-GP4-GP5 were primed for significant higher levels of anti-PRRSV CTL responses than mice immunized with rAd-GP3 and rAd-GP5. These findings suggested that the recombinant adenoviruses expressing fusion proteins GP3-GP5 or GP3-GP4-GP5 might be an attractive candidate vaccine for preventing PRRSV infection.     

Permeabilization of the plasma membrane by Ebola virus GP2

The glycoprotein (GP) of Ebola virus (EBOV) is a multifunctional protein known to play a role in virus attachment and entry, cell rounding and cytotoxicity, down-regulation of host surface proteins, and enhancement of virus assembly and budding. EBOV GP is synthesized as a precursor which is subsequently cleaved to yield two disulfide-linked subunits: GP1 (surface-exposed [SU] subunit) and GP2 (membrane-anchored [TM] subunit). We sought to determine the effect of membrane-anchored GP2 protein expression on the integrity of host cell lipid membranes. Our findings indicated that: (i) expression of GP2 enhanced membrane permeability to hygromycin-B (hyg-B), (ii) the transmembrane (TM) domain of GP2 was essential for enhanced membrane permeability, (iii) amino acids (aa) 667ALF669 within the TM region of GP2 were important for enhanced membrane permeability, and (iv) EBOV infected cells were more permeable to hyg-B than mock infected cells. Together, these data suggest that the TM region of GP2 modifies the permeability of the plasma membrane. These findings may have important implications for GP-induced cell damage and pathogenesis of EBOV infection.     

Antibodies to Hepatitis A Virus in Immune Serum Globulin

Two hundred one immune serum globulin (ISG) lots manufactured in the US between 1967 and 1977 were tested for antibodies to the hepatitis A virus (anti-HAV) by a competitive-inhibition radioimmunoassay (RIA); a lesser number were also tested by immune adherence hemagglutination (IAHA). The percentage of ISG lots that contained anti-HAV with a titer of 1:100 or greater by RIA was 50% for those manufactured in 1967, 69% for those manufactured in 1972, and 100% for those manufactured in 1977. The percentage of lots with anti-HAV titers equal to or greater than 1:500 by RIA was 7% in 1967, 18% in 1972, and 70% in 1977. Only ten lots of ISG (5%) had anti-HAV titers of 1:1,000 or greater by RIA; seven of these were manufactured in 1977. Both the mean titer of anti-HAV in ISG lots and the percentage of lots containing significant titers of this antibody appear to have increased in the US over the past ten years. This may reflect the increased use of source plasma from paid plasmapheresis donors in the US during this period. The lower titers of anti-HAV in the older lots of ISG studied were shown not to be due to fragmentation of antibody molecules during storage.     

Lack of evidence for an association between seminoma and human papillomavirus infection using GP5+/GP6+ consensus primers

Testicular germ cell tumors account for about 1% of all cancers. The incidence of these tumors is increasing and they represent the most common solid malignancies of young men aged 15–40 years with seminoma being one of the most common histotype. Pathogenesis of testicular germ cell tumors remains unknown and, although cryptorchidism is considered the main risk factor, there is evidence of an association with environmental and genetic risk factors. Human papillomaviruses (HPV) are a family of DNA viruses and represent a major risk factor for cervical cancer. In addition, they have been associated with other human non‐malignant and malignant diseases, including breast and head and neck cancer. HPV sequences have been detected throughout the male lower genitourinary tract as well as in seminal fluid and an increased testicular tumorigenesis has been reported in HPV transgenic mice. Aim of this study was to evaluate the potential involvement of HPV in human testicular tumorigenesis. Real‐time PCR employing GP5+/GP6+ consensus HPV primers was used to examine the presence of HPV sequences in a subset of human seminoma (n = 61) and normal testicles (n = 23). None of the specimens tested displayed the presence of HPV DNA. These findings do not support an association between HPV and human seminoma and warrant further studies to assess definitively the role of these viruses in human testicular tumorigenesis. J. Med. Virol. 85:105–109, 2012. © 2012 Wiley Periodicals, Inc.     

中国狂犬病毒疫苗株（5aG）糖蛋白基因在痘苗病毒中的表达及免疫效果观察

将克隆到的中国狂犬病毒疫苗株（５ａＧ）的糖蛋白基因重组到痘苗病毒ＴＫ区，并在痘苗病毒Ｐ１１启动子的控制下，构建了狂犬－痘苗重组病毒（ＶＶａＧ）。经间接免疫荧光和Ｗｅｓｔｅｒｎ免疫印染证明，重组病毒ＶＶａＧ能良好地表达狂犬病毒糖蛋白，其分子量约为６６００。用ＶＶａＧ免疫小鼠，７ｄ便可诱生较高的狂犬病毒中和抗体，２１ｄ达４１６９，并能１００％保护狂犬病毒本毒株和国际标准攻击毒（ＣＶＳ）的致死量攻击。     

血小板特异性抗体及HLA抗体引起血小板输注无效的研究

目的分析血小板输注无效（PTR）患者血小板同种抗体,并对其HPA及HLA-Ⅰ类抗原进行基因分型,探讨血小板输注无效与血小板同种抗原／HLA—Ⅰ类抗原相关性。方法应用ELISA方法对17例血小板输注无效患者血清中的血小板抗体进行检测;运用PCR—SSP方法,采用HPA分型试剂盒检测血小板同种抗原7个抗原系统HPA-1、2、3、4、5、6、15,以及HLA分型试剂盒对HLA—A／B抗原进行基因分型。结果6名患者单独表达HLA抗体,4名患者表达血小板特异性糖蛋白抗体,3例HLA抗体和血小板特异性糖蛋白抗体共同表达．其中以GPⅡb／Ⅲa为主。对17例患者HPA系统和HLA-Ⅰ类抗原基因分型,发现HPA-3系统中a的基因频率高达0．676,b为0．324;HLA—A＊02、HLA—A＊24、HLA—A＊11和HLA—B＊60、HLA—B＊13、HLA—B＊46多见。结论HLA抗体和血小板特异性糖蛋白抗体表达引起血小板输注无效,了解胛R与HPA／HLA—Ⅰ类抗原的相关性对指导临床血小板配合性输注具有重要意义。     

Enhanced immunogenicity of the modified GP5 of porcine reproductive and respiratory syndrome virus

The ORF5-encoded major envelope glycoprotein (GP5) is one of the key immunogenic proteins of the porcine reproductive and respiratory syndrome virus (PRRSV) and is the leading target for the development of the new generation of vaccines against PRRS. However, weak and tardy neutralizing antibodies have been elicited in several developed experimental vaccines expressing PRRSV GP5. More recent evidence has demonstrated a non-neutralizing decoy epitope upstream of the neutralizing epitope of GP5, which might prevent the development of a strong neutralizing antibody response against PRRSV. In the present study, we modified the ORF5 gene by inserting a Pan DR T-helper cell epitope (PADRE) between the neutralizing epitope and the decoy epitope to minimize or eliminate the decoy effect of the non-neutralizing epitope. The immunogenicity of the modified GP5 was further evaluated using DNA vaccination. The results showed that significantly enhanced neutralizing antibodies were elicited in mice immunized with the DNA construct expressing the modified GP5 compared with the native GP5. Slightly increased levels of GP5-specific ELISA antibodies and T-cell proliferative activities were also observed. These results indicate that the high immunogenicity of the modified GP5 might facilitate the development of improved PRRS vaccines in the future.     

Generation and immunogenicity of transgenic potato expressing the GP5 protein of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that has caused huge economic losses in the global swine industry. The advent of molecular farming has provided a cost-effective strategy for the development of transgenic plants as bioreactors to produce recombinant proteins. In this study, transgenic potato expressing GP5 protein of PRRSV was produced by Agrobacterium-mediated transformation, and confirmed using Southern blot and RT-PCR analyses. Recombinant GP5 protein was detected by ELISA and Western blot analyses. Mice immunized with transgenic potato extracts generated both serum and gut mucosal-specific antibodies, although low levels of neutralizing antibodies were elicited. This study provides a new approach for the production of vaccines against PRRSV.     

NP9对鼻咽癌细胞系CNE1基因表达谱的影响

目的： 确定NP9表达对鼻咽癌CNE1细胞基因表达谱的影响。方法: 稳定表达NP9基因和稳定转染空载体的CNE1细胞分别作为基因芯片分析的实验组和对照组，用高通量的基因芯片筛选实验组细胞的差异表达基因，荧光定量逆转录PCR验证部分基因表达差异。结果: 所分析的14 500个基因中，266个检测出有表达差异，其中82个基因呈现表达上调（RA＞1），184个基因显示表达下调（RA＜1）。显著上调和下调的基因分别为34个（RA＞1.5）和75个（RA＜1.5）。结论: NP9基因的表达导致了CNE1细胞中与细胞周期调控、细胞增殖和分化、细胞信号转导、细胞黏附相关基因表达的改变，为NP9的功能及分子机制研究提供了新的线索。