在大肠杆菌中分别表达汉滩病毒素膜糖蛋白G1和G2

利用PCR方法扩增了汉滩病毒76－118株囊膜糖蛋白G1和G2的编码区基因,并将PCR产物克隆到T-载体中,用限制性内切酶将G1和G2的编码区基因切下,并克隆到表达载体pBV220中构建G1和G2的表达质粒。诱导表达后在SDS－PAGE凝胶中未见表达产物带,表达的G1和G2能与部分抗G1和G2的单克隆抗体发生反应,但用Western－blot方法不能检测到表达产物。用表达的G1和G2免疫小白鼠能刺激小白鼠产生特异性抗汉摊病毒的抗体,间接免疫荧光抗体的滴度可分别达到1:160和1：320。     

用不同载体在大肠杆菌中表达汉滩病毒囊膜糖蛋白G1和G2

为提高汉瘫病毒囊膜糖蛋白G1和G2的表达水平,利用两种不同的表达载体pKK223－3和pGEX－4T－1构建G1和G2的表达质粒,其中PGEX－4T－1为融合蛋白表达载体。诱导所构建的G1和G2表达质粒表达G1和G2,用表达的G1和G2免疫小白鼠诱导产生的抗汉瘫病毒特异性的间接免疫荧光抗体摘度无明显差别,从而说明表达载体对汉瘫病毒G1和G2在大肠杆菌中的表达水平影响不大,表达水平都较低。同时,利用PGEX－4T－1构建G1和G2的部分多肽的表达质粒,但表达水平较完整的G1和G2无明显提高。     

庚型肝炎病毒包膜糖蛋白E2基因在昆虫细胞中的表达

用ＰＣＲ扩增出ＨＧＶＥ２全基因,克隆进杆状病毒表达载体ｐＦＡＳＴＢＡＣＨＴａ中,构建成重组转座载体ｐＦＡＳＴＢＡＣＥ２,转化ＤＨ１０ＢＡＣ大肠杆菌感受态细胞,筛选阳性菌落,抽提大分子质粒ＤＮＡ,获得含ＨＧＶＥ２基因的重组杆状病毒穿梭载体,转染昆虫草地夜蛾Ｓｆ９细胞,出现细胞病变后,收集含有重组病毒颗粒的培养上清,重新感染草地夜蛾Ｓｆ９单层细胞及甜菜夜蛾幼虫,分别收集Ｓｆ９细胞和甜菜夜蛾幼虫体内的血淋巴细胞,进行１２％ＳＤＳ聚丙烯酰胺凝胶电泳,可见表达的融合蛋白带,经亲和层析进行蛋白纯化,用ＥＬＩＳＡ方法检测各类血清标本,初步研究ＨＧＶＥ２糖蛋白的抗原性     

Mechanisms of HIV envelope-induced T lymphocyte apoptosis

Infection by the human immunodeficiency virus (HIV) is characterized by a progressive depletion of CD4 T lymphocytes, which leads to dysfunction of the immune system. Although a variety of mechanisms may contribute to the gradual T cell decline that occurs in HIV-infected patients, abnormal apoptosis of infected or bystander T lymphocytes is an important event leading to immunodeficiency. The HIV envelope glycoprotein plays a crucial role in HIV associated apoptosis through both death receptor-mediated and mitochondria-dependent pathways. This review summarizes current knowledge of Env-mediated T lymphocyte apoptosis.     

Mechanisms of HIV envelope-induced T lymphocyte apoptosis

Infection by the human immunodeficiency virus (HIV) is characterized by a progressive depletion of CD4 T lymphocytes, which leads to dysfunction of the immune system. Although a variety of mechanisms may contribute to the gradual T cell decline that occurs in HIV-infected patients, abnormal apoptosis of infected or bystander T lymphocytes is an important event leading to immunodeficiency. The HIV envelope glycoprotein plays a crucial role in HIV associated apoptosis through both death receptor-mediated and ...     

单纯疱疹病毒2型糖蛋白D成熟肽基因毕赤酵母表达载体的构建及序列分析

构建单纯疱疹病毒2型包膜糖蛋白D成熟肽基因毕赤酵母表达载体,并对序列进行分析,为进行高抗原性的真核表达重组gD蛋白奠定基础。采用PCR扩增HSV2-gD成熟肽基因,将该段基因克隆于pGEM-T克隆载体,转化鉴定后,与巴斯德毕赤酵母表达载体(pPIC9K)酶切连接,转化大肠杆菌DH5α,筛选测序确定构建了pPIC9K?gD的真核表达载体,对克隆的序列进行分析,预测表达产物的理化特性及抗原性。结果显示,获得的重组的酵母表达载体pPIC9K-gD,测序结果证实为HSV2-gD成熟肽基因,序列分析其高度保守,预测蛋白分子量40.63kD,等电点pI为7.15,包含完整成熟肽分值达1.7的多个抗原决定簇。成功构建了HSV2-gD成熟肽基因的毕赤酵母表达载体。     

猪瘟病毒强弱毒株和野毒株E2全基因序列测定及比较分析

为了比较猪瘟病毒 (HCV)野毒株、疫苗株及标准株之间E2基因抗原区域的差异 ,采用RT PCR扩增了HCV石门株、兔化弱毒疫苗株、野毒 0 3及 0 7株的囊膜糖蛋白E2 (gp55)全基因的cDNA片段 ,分别克隆于pGEM T载体中并对其进行了核苷酸序列测定及氨基酸序列的推导 ,同时进行了同源性比较及E2结构与功能的分析。所测 4株HCVE2基因的长度均为1 2 73bp,所编码的氨基酸序列均包括部分信号肽序列和完整的跨膜区序列 ,共由 381个氨基酸组成 ;4个毒株E2蛋白N末端的 683位至 690位信号肽序列 (WLLLVTGA)和C末端 1 0 30～1 0 63位跨膜区均为保守序列 ,而且具疏水性 ;N末端抗原功能区中 ,4个E2蛋白与其它所比较序列在位于第 753位至 759位氨基酸处 ,均有一段保守序列RYLASLH ,无一氨基酸发生变异 ,为亲水性 ,在整个E2蛋白抗原谱中抗原性峰值为最高 ,推测对抗原性产生起重要作用 ;4个E2蛋白的氨基酸序列中均含有 1 5个半胱氨酸 (Cys)残基 ,其数量及位置与国外五株HCV(Brescia ,C ,Alfort.ALD和GPE)完全一致。表明…     

风疹病毒包膜糖蛋白E1中二硫键对其细胞融合活性的影响

为了探讨风疹病毒包膜糖蛋白E1中二硫键对风疹病毒细胞融合活性的影响,在构建重组载体pBSK-SPE2E1的基础上,利用PCR定点突变与体内同源重组相结合的方法,构建了11个突变体,分别将E1外功能区的11个半胱氨酸残基突变为其它氨基酸残基,从而去除一个二硫键,利用Giemsa染色法定性检测由此引起的细胞融合情况,流式细胞术检测导入的外源DNA在细胞表面的表达效率,血吸附检测重组表达的突变体蛋白的受体识别活性。结果表明E1外功能区的10个二硫键对RV的细胞融合活性都有重要影响,任何一个二硫键的去除均导致E1的细胞融合活性丧失;其中第5和第8个半胱氨酸残基所形成的二硫键与E2和E1的相互作用有关,第3、第4和第13个半胱氨酸残基所形成的二硫键可能直接影响E1的细胞融合功能。     

Immunization with cytomegalovirus envelope glycoprotein M and glycoprotein N DNA vaccines can provide mice with complete protection against a lethal murine cytomegalovirus challenge

Human cytomegalovirus virions contain three major glycoprotein complexes (gC I, II, III), all of which are required for CMV infectivity. These complexes also represent major antigenic targets for anti-viral immune responses. The gC II complex consists of two glycoproteins, gM and gN. In the current study, DNA vaccines expressing the murine cytomegalovirus (MCMV) homologs of the gM and gN proteins were evaluated for protection against lethal MCMV infection in a mouse model. Humoral and cellular immune responses, spleen viral titers, and mice survival and body-weight changes were examined. The results showed that immunization with gM or gN DNA vaccine alone was not able to offer good protection, whereas co-immunization with both gM and gN induced an effective neutralizing antibody response and cellular immune response, and provided mice with complete protection against a lethal MCMV challenge. This study provides the first in vivo evidence that the gC II (gM-gN) complex may be able to serve as a protective subunit antigen for future HCMV vaccine development.     

12株猪瘟病毒E2基因主要抗原区域的序列差异分析

用RTPCR扩增了12个不同时期分离的HCV毒株E2基因主要抗原区域的cDNA片段并对其进行了序列测定。应用DNAstar序列分析软件对所测的12个HCV毒株与国内外已知的6个毒株Alfort株、Ald株、Brescia株、Gpe株、C株、CW株及早期已测定的HCLV株、HCVSM株和北京顺义株(BJSY2/96)3个毒株的相应片段进行了同源性比较分析。E2基因主要区域长度均为224 bp,包括从HCV 2485到2708位的E2基因B、C区域。所测的疫苗株HCLV与国外测得的疫苗株C株核苷酸及氨基酸同源性分别为991%和100%,表明目前应用的疫苗株是稳定的;用目前我国流行的部分野毒株对HCLV株免疫猪的攻击试验表明,HCLV对野毒株均具有很好的免疫力,这与序列分析结果相吻合。根据系统树分析,可将HCV分为两大群,5株90年代的野毒株及1株80年代的野毒株(其中北京3株、河南2株、广东1株)均与国内外C株、标准株属同一群(即第一群),其核苷酸及氨基酸的同源性分别为857%～100%和838%～100%;与Alfort株同属第二群的有6个野毒株(广西北海、辽宁、河北黄骅、吉林、深圳光明、四川成都),其中80年代与90年代的野毒株各有三株,核苷酸及氨基酸的同源性分别为843%～100%和851%～100%;21株HCV的核苷酸及氨基酸的同源性分别为781%～100%和784%～100%。两群之间的特征性差异表现在713和729位氨基酸位点的不同,经分析发现猪瘟野毒株具有复杂性与多样性。     

汉滩病毒包膜糖蛋白糖基化位点突变体重组假病毒的构建及初步鉴定

目的：为进一步研究汉坦病毒包膜糖蛋白的糖基化与病毒的感染性和免疫原性等的关系,构建含有汉滩病毒（HTNV）囊膜糖蛋白（GP）糖基化位点突变体的重组假病毒。方法：利用定点突变的方法,分别突变了HTNV 76-118株的5个N-糖基化位点并克隆入慢病毒表达载体,与包装质粒共转染293T细胞,构建5株重组假病毒。感染HEK293细胞后,进行RT-PCR鉴定及免疫荧光检测。结果：经测序显示构建的含有N-糖基化位点突变体的5个重组假病毒原序列中的天冬酰胺（N）均被置换为谷氨酰胺（Q）。RT-PCR结果显示5个重组假病毒均有HTNV GP基因的表达。免疫荧光检测5个重组假病毒均可表达HTNV的Gn和Gc蛋白。结论：成功构建了含有HTNV包膜糖蛋白糖基化位点突变体的5个重组假病毒,分别命名为rLV-M1、rLV-M2、rLV-M3、rLV-M4和rLV-M5。本研究为明确N-糖基化对汉坦病毒生物学活性的影响提供了有利的研究工具,并为汉坦病毒疫苗及致病机理的进一步研究打下了一定的基础。     

Changes in prevalence of herpes simplex virus type 1 and 2 antibodies from 1973 to 1993 in the rural districts of Japan

Using the gG-capture ELISA, changes in the seroprevalence of HSV-1 and HSV-2 from 1973 to 1993 were studied for 614 sera collected from general adults living in rural Japan. The HSV-1 seroprevalence for men and women decreased from 75.3 and 80.6% in 1973 to 54.4 and 59.6%, respectively, in 1993. The HSV-2 seroprevalence also decreased from 10.2 and 9.9% in 1973 to 1.8 and 1.2%, respectively, in 1993. Although the decrease in HSV-2 prevalence seemed to be correlated with the general decrease of sexually transmitted diseases in Japan since the 1950s, these findings should not be interpreted as typical, as HSV-2 infections are particularly known to distribute unevenly among populations, according to sexual activity and cohorts.     

北京地区人偏肺病毒膜表面糖蛋白G编码基因和特征的分析

为了解北京地区人偏肺病毒(hMPV)膜表面糖蛋白G编码基因的特征,提取2003年和2004年各6份hMPV阳性的临床呼吸道标本中的RNA,经用随机引物合成cDNA后,用特异性引物扩增G蛋白全基因,克隆至pBS-T载体中并进行测序。用生物软件与GenBank中hMPV的基因序列进行比较和种系进化分析。12株hMPV可以分为两个主要的进化簇1和2。每个进化簇还可以再分为不同的亚进化簇。3株(2003年)hMPV属于进化簇1;9株(3株2003年和6株2004年)hMPV属于进化簇2。这12株hMPV G蛋白基因的核苷酸长度在624~711nt,使用了两种不同的终止密码,编码的氨基酸长度为208~236 aa,蛋白质分子量为22.9~25.8kD。与进化簇2相比,进化簇1的3株hMPV都出现了3个核苷酸的缺失,但未改变读码框架。同一进化簇内的hMPV G蛋白基因核苷酸和氨基酸的同源性分别为95.7%~100%和91.8%~100%,不同进化簇间的核苷酸和氨基酸的同源性分别为56.3%~57.4%和34.3%~38.2%。疏水性分析表明这12株hMPV G蛋白是典型的Ⅱ型跨膜锚定蛋白,分别与两个进化簇代表株的疏水图一致。这些hMPV的G蛋白氨基酸的替换集中在胞外区。它们都含有高百分比的脯氨酸(P)、丝氨酸(S)和苏氨酸(T),含有相当数量的位于胞外区的O-连接糖基化位点。这些hMPV的G蛋白的N-连接糖基化位点数目和位置不尽相同。通过对G蛋白基因的分析显示,2003年和2004年北京地区流行的hMPV分属于两种不同的进化簇。基因分析显示hMPV的G蛋白是高糖基化的膜表面蛋白,具有与同属于副粘病毒科、肺病毒亚科的人呼吸道合胞病毒(hRSV)的G蛋白相似的基因特征,推测其功能和在感染免疫中的作用相当于hRSV的G蛋白,但有待进一步深入研究予以证实。     

Prevalence of Herpes Simplex Virus Type 1- and 2- Specific Antibodies among the Acute,Recurrent, and Provoked Types of Female Genital Herpes

Sixty-eight sera from the acute, recurrent, and provoked types of female genital herpes were compared for the seroprevalence of herpes simplex virus (HSV) types 1 and 2 by immunodot assay using HSV glycoprotein G. In the HSV-1-isolated patients, no HSV-2 antibodies were detected, whereas in the HSV-2-isolated patients, HSV-1 seroprevalence was 9% for the acute type, 89% for the provoked type (P< 0.005), and 55% for the recurrent type (P<0.05). The natural history of female genital herpes and the possible protective role of pre-existing antibodies in preventing the acquisition or clinical manifestation of a subsequent HSV infection are discussed.     

我国猪瘟病毒兔化弱毒株囊膜糖蛋白E0基因的克隆及序列测定

采用异硫氰酸胍一步法从４８０代猪瘟病毒兔化弱毒株（ＨＣＬＶ）脾毒中提取总ＲＮＡ,以该ＲＮＡ为模板,进行反转录,然后采用套式ＰＣＲ扩增出ＨＣＬＶ的囊膜糖蛋白Ｅ０基因,琼脂糖凝胶电泳表明其大小与预计相符。将扩增出的Ｅ０基因克隆到ｐＧＥＭＴ载体中,用自动序列分析仪对其进行序列测定。将测得的序列及推导的氨基酸序列与国外测得的Ｃ株相应序列进行比较,结果发现,它们之间核苷酸序列同源性为９９．０８％,氨基酸序列同源性为９８．４２％。     

基因治疗慢病毒载体的转导增强策略*

基于慢病毒载体的体外基因治疗已在临床试验中取得良好的效果,有望治愈一些造血系统的单基因遗传病。通过提高靶细胞转导效率和减少转导中病毒载体量,基因治疗有效性、安全性和成本都可以得到改善。不同包膜糖蛋白伪型慢病毒载体通过与细胞膜表面的不同受体结合,促进病毒黏附和入胞,增强不同靶细胞的病毒转导效率。此外病毒转导增强剂可以在病毒进入细胞过程或进入后发挥作用,在提高转导效率的同时使靶转导基因在体内长期稳定表达。通过对这两类方法的总结回顾,旨在为慢病毒载体的转导效率提供新的优化策略,使基因治疗得到更广泛的应用。     

Expression of a human cytomegalovirus gp58 antigenic domain fused to the hepatitis B virus nucleocapsid protein

Abstract Hepatitis B virus core antigen (HBcAg) has been used as a carrier for expression and presentation of a variety of heterologous viral epitopes in particulate form. The aim of this study was to produce hybrid antigens comprising HBcAg and an immunogenic epitope of human cytomegalovirus (HCMV). A direct comparison was made of amino and carboxyl terminal fusions in order to investigate the influence of position of the foreign epitope on hybrid core particle formation, antigenicity and immunogenicity. HCMV DNA encoding a neutralising epitope of the surface glycoprotein gp58 was either inserted at the amino terminus or fused to the truncated carboxyl terminus of HBcAg and expressed in Escherichia coli . The carboxyl terminal fusion (HBc_3–144-HCMV) was expressed at high levels and assembled into core like particles resembling native HBcAg. Protein with a similar fusion at the amino terminus (HCMV-HBc_1–183) could not be purified or characterised immunologically, although it formed core like particles. HBc_3–144-HCMV displayed HBc antigenicity but HCMV antigenicity could not be detected by radioimmunoassay or western blotting using anti-HCMV monoclonal antibody 7–17 or an anti-HCMV human polyclonal antiserum. Following immunisation of rabbits with HBc_3–144-HCMV, a high titre of anti-HBc specific antibody was produced along with lower titres of HCMV/gp58 specific antibody.     

Production of recombinant gG-1 protein of herpes simplex virus type 1 in a prokaryotic system in order to develop a type-specific enzyme-linked immunosorbent assay kit

The herpes simplex viruses are important causes of disease worldwide. Herpes simplex virus type 1 (HSV-1) is the primary cause of oral-facial and pharyngeal infections and may cause herpetic whitlow, eye infections as well as severe and sometimes dangerous infections of the eyes and brain. HSV-1 also accounts for 10-15% of all genital herpetic infections. Therefore, laboratory diagnosis of this virus and development of diagnostic serological techniques for HSV-1 is of particular importance. In the present study, pTrc His2A-gG1 plasmid, containing the full-length glycoprotein G (gG) protein, was produced in a prokaryotic system for the first time. Upon confirmation of a 37-kDa gG-1 protein production in a prokaryotic system based on western blotting and monoclonal antibodies, the protein was produced at a large scale and purified by ion-exchange chromatography using DEAE-sepharose. An HSV-1 type-specific diagnostic kit was designed and developed and the specificity and sensitivity of this kit were demonstrated to be 89.5% and 100%, respectively, as compared with a commercially available kit. A significant correlation was shown between the developed kit and the commercial kit.     

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。     

Effect of the infectious laryngotracheitis virus (ILTV) glycoprotein G on virus attachment,penetration, growth curve and direct cell-to-cell spread

The secreted alphaherpesvirus glycoprotein G (gG) works differently from other proteins. Analysis of the role of ILTV gG in virus attachment, penetration, direct cell-to-cell spread (CTCS) and the growth curve showed that gG or its antibody had no effect on ILTV attachment and penetration and that the gG antibody reduced the virus plaque size and the one-step growth curve on chicken embryo liver (CEL) cells, but gG did not affect the virus plaque size or the one-step growth curve on CEL cells. Laser scanning confocal microscopy (LSCM) detection showed that ILTV gG is located in the perinuclear region and the membrane of the CEL cells. These results suggested that ILTV gG might contribute to direct cell-to-cell transmission.