乙型肝炎病毒基因疫苗联合抗原蛋白免疫效应的评价

目的探讨乙型肝炎病毒(HBV)基因疫苗联合乙型肝炎病毒表面抗原S蛋白(HBsAg)免疫Balb/c小鼠的效应.方法构建编码S蛋白的重组真核表达质粒pCR3.1-S作为HBV基因疫苗.以HBV基因疫苗联合HBsAg蛋白免疫接种Balb/c小鼠,同时以HBV基因疫苗或S蛋白单独接种Balb/c小鼠作为对照.采用ELISA法检测免疫小鼠的抗HBs应答,3H-TdR掺入法检测免疫小鼠的淋巴细胞增殖反应.结果免疫接种2周、4周后,联合免疫组抗HBs滴度高于HBV基因疫苗单独接种组,但低于S蛋白单独接种组;6周后,HBV基因疫苗组抗HBs滴度最高,联合组次之.各组间免疫小鼠的淋巴细胞增殖反应无显著差异性(P＞0.05).结论 HBV基因疫苗联合S蛋白免疫Balb/c小鼠无优势.     

登革Ⅱ型病毒感染Balb/C实验小鼠的研究

目的：探讨Balb/C小鼠作为实验动物模型感染登革病毒的情况。方法：小鼠腹部皮下多点注射接种大剂量登革病毒后在一定时间内检测血清病毒,抗体及腹腔巨噬细胞,脾细胞登革病毒感染情况。结果：Balb/C实验小鼠接种感染登革病毒以后能产生1-2d病毒血症,接种后第4d血清中即可检测到特异性抗体,说明在登革病毒感染早期即产生可检测到的特异性抗体,而腹腔巨噬细胞在接种后前1-2d登革病毒感染率较高,但下降很快,第4d以后感染水平很低,小鼠脾细胞登革病毒感染率极低,几乎检测不到阳性,结论：Balb/C实验小鼠可以用于实验感染登革Ⅱ型病毒。     

639例正常人群中HBV—pre—S2检测结果分析

乙型肝炎是流行十分之泛的传染病,早已引人瞩目。目前诊断乙型肝炎主要依据检测 HBV 的抗原抗体。由于乙肝病毒(HBV)外膜 HBsAg 由 Pre—S_1、Pre—S_2及 S 蛋白组成,其中 Pre—S_2蛋白含有能够结合多聚人血清白蛋白受体可能同病毒与肝细胞     

MUC1基因疫苗对乳腺肿瘤抑制的实验研究

目的:研究人粘蛋白MUC1基因DNA疫苗诱导小鼠体内免疫应答及对乳腺肿瘤的特异性抑制作用。方法:采用股四头肌注射法接种PcDNA3.1(+)-MUC1质粒,通过ELISA法检测小鼠血清MUC1抗体、脾细胞产生IL-2和IFN-γ的量及血清中TNF水平,通过乳酸脱氢酶释放法测定CTL对MCF-7细胞的杀伤活性。结果:经PcDNA3.1(+)-MUC1免疫小鼠后脾细胞分泌IL-2、IFN-γ及血清TNF水平较对照组明显增高,差异有统计学意义;在效靶比为100∶1、50∶1、25∶1、12.5∶1时,MUC1基因疫苗免疫组小鼠CTL对人乳腺癌细胞系MCF-7杀伤率分别为63.8%、51.2%、43.5%、22.5%,较空载体PcDNA3.1(+)组小鼠和灭菌生理盐水组小鼠均明显升高,差异有统计学意义(P<0.05)。结论:MUC1基因DNA疫苗能够激活小鼠Th1细胞,诱导小鼠产生抗MUCl特异性抗体,诱导产生杀伤高表达MUC1基因的乳腺癌细胞的CTL,为MUC1基因疫苗用于乳腺肿瘤生物治疗提供了一定的实验依据。     

重组体pEF—HCF诱发小鼠体液免疫反应的研究

目的：探讨基因疫苗在HCV感染中的预防和治疗价值,方法：将HCV C区基因插入pEF质粒EF－1α启动子下游。构建pEF-HCV C重组质粒,然后经肌肉注射及皮下注射免疫Balb/c小鼠。结果：在接种后3周内在肌注组内检测到抗HCV,但是抗体滴度较低,在加强免疫后7天血清抗－HCV明显增高,而其它几组 未检出抗-HCV。结论：构建的HCVDNA重组体可诱发Balb/c上鼠产生体液免疫应答。     

丙型肝炎病毒E2蛋白DNA疫苗诱导小鼠产生中和抗体的研究

目的探讨丙型肝炎病毒(HCV)包膜E2蛋白DNA疫苗诱导小鼠产生中和抗体的可行性。方法构建截除疏水性羧基末端的HCV包膜蛋白表达质粒pCI-1b661以及同时截除疏水性羧基末端和高变区1(HVR1)的表达质粒pCI-1b661Δ,转染293T细胞,以Western blot和ELISA检测细胞内和培养上清中的HCVE2蛋白,将两种表达质粒及空载体分别肌注免疫BALB/c小鼠,以ELISA检测小鼠血清中的HVR1抗体,以HCV假病毒颗粒(HCVpp)分析小鼠血清的中和活性。结果 2种表达质粒均能表达分泌性截短型E2蛋白。pCI-1b661免疫的8只小鼠血清中均可检测到HVR1抗体,而pCI-1b661Δ免疫血清中未检测到HVR1抗体。pCI-1b661和pCI-1b661Δ免疫血清对HCVpp的中和率分别为(78.5±13.8)%和(38.7±6.5)%,差异有显著性(P<0.01)。pCI-1b661免疫组小鼠血清的中和率与HVR1抗体水平呈正相关(r=0.967,P<0.01)。结论表达截短型E2蛋白的DNA疫苗能诱导产生HCV中和抗体,其主要成员为HVR1抗体。     

免疫接种乙型肝炎病毒外膜小S蛋白产生的细胞免疫反应

目的　研究BALB/c(H 2 d)小鼠免疫接种乙型肝炎病毒 (HBV)外膜小S蛋白 (HBVsmallSenvelopeprotein,S HBsAg)产生的细胞免疫反应。方法　BALB/c(H 2 d)小鼠腹腔接种 0～ 5 μg的S HBsAg ;4周后分离脾淋巴细胞 ,体外分别用特异性多肽、S HBsAg刺激 ,分别用51Cr释放实验3 H掺入实验检测细胞毒T淋巴细胞 (cy totoxicTlymphocyte,CTL)及辅助T淋巴细胞 (helperTlymphocyte,HTL)增殖实验。结果　CTL反应的靶细胞自然释放率 ,自然释放 /最大释放 ,平均数±标准差为 (2 8 76± 13 4 5 ) % ;分别接种 0 ,0 6 5 ,1 2 5 ,2 5 ,5 μgS HB sAg的BALB/c(H 2 d)小鼠脾淋巴细胞CTL特异性释放率 (平均数±标准差 )分别为 (31 2 1± 9 2 3) % ,(4 2 36±19 32 ) % ,(91 2 1± 2 2 97) % ,(6 9 2 5± 2 4 l3) % ,(5 1 4 9± 2 1 6 6 ) % ,均高于作为对照的 0组小鼠脾淋巴细胞CTL释放率 ,差异有统计学意义 (P <0 0 5 ) ;HTL增殖实验 ,用S HBsAg刺激的 0 6 5～ 5 μg实验组BALB/c(H 2 d)小鼠脾淋巴细胞cpm值 (平均数±标准差 )分别为 3830 2 4± 14 96 12 ,2 736 19±12 38 0 6 ,4 10 0 37±172 3 74 ,12 4 6 18± 10 88 88,均高于未用S HBsAg刺激的对照组BALB/c(H 2 d)小鼠脾淋巴细胞cpm值 ,差异有统计学意义 (P     

小鼠免疫接种乙型肝炎病毒表面S疫苗引起的细胞毒T淋巴细胞反应

目的　研究小鼠免疫接种乙型肝炎病毒 (HBV)表面 S抗原疫苗 (S- HBs Ag)后引起的特异性、MHC- 限制性细胞毒 T淋巴细胞 (cytotoxic T lymphocytes,CTL)反应。方法　分别给 BAL B/c(H- 2 d)小鼠腹腔内接种 0～ 6 μg S- HBs Ag,2周后加强 1次 ,4周后分离小鼠脾淋巴 T细胞 ,体外用小鼠 Ld限制的、S- HBs Ag特异性 CTL表位多肽 S2 8- 39- 刺激 ;用特异性多肽 S2 8- 39- 、51 Cr标记的 P815细胞作为靶细胞 ;4 h51 Cr释放实验检测 CTL 反应。结果　 0、0 .6 5、1.2 5、2 .5、5 μg组小鼠 ,CTL 特异性释放率分别为 31.2 1%± 9.2 3%、4 2 .36 %± 19.32 %、91.2 1%± 2 2 .97%、6 9.2 5 %± 2 4 .13%、5 1.4 9%± 2 1.6 6 1% ;只接受 1次免疫接种的小鼠 ,其脾淋巴细胞特异性 CTL 特异性释放率分别为 2 7.34%± 14 .2 5 %、32 .2 7%± 15 .35 %、5 6 .2 8%± 2 4 .35 %、4 4 .34%± 18.6 5 %、4 0 .76 %±5 6 %。结论　 BAL B/c(H- 2 d )小鼠腹腔内接种 S- HBs Ag引起特异性 CTL 反应 ,加强免疫能够提高 CTL 反应。     

前S2抗原与乙型肝炎病毒复制关系的探讨

乙肝病毒(HBV)血清标志物检测是用于判断乙肝病人免疫反应状态的重要方法。乙肝病毒定量检测是衡量HBV感染复制水平有价值的标准。两者共同反映了病毒的复制的特征。近年来对HBV外膜的研究日趋深入,HBV外膜蛋白即HBsAg,分为S基因、前S1区段和前S2区段。经研究发现前S蛋白(PreS)     

人巨细胞病毒IE1核酸疫苗的免疫效果研究

[目的]用人巨细胞病毒(HCMV)IE1核酸疫苗pcDNA3.1(-)-IE1免疫BALB/c小鼠,初步研究其产生的体液免疫和细胞免疫应答水平。[方法]将pcDNA3.1(-)-IE1通过肌肉注射免疫BALB/c小鼠,通过PCR测定和免疫组化检测其在肌细胞中的表达,细胞因子测定、淋巴细胞转化实验检测免疫效果。[结果]PCR检测到与IE1大小一致的片段,免疫组化结果显示IE1基因在小鼠肌细胞中表达IE1目的蛋白。小鼠脾淋巴细胞经PHA刺激后,实验组IL-4、IL-2、IFN-γ含量以及淋巴细胞转化率显著增高,与对照组比较差异有统计学意义(P﹤0.05)。[结论]pcDNA3.1(-)-IE1核酸疫苗能在BALB/c小鼠肌细胞中稳定存在并能表达HCMV IE1蛋白,pcDNA3.1(-)-IE1核酸疫苗可诱导BALB/c小鼠脾细胞分泌IL-4、IL-2、IFN-r并刺激BALB/c小鼠脾细胞增殖。     

Control of bovine virus diarrhoea virus

Bovine virus diarrhoea (BVD) virus is ubiquitous in cattle populations throughout the world. Strategies for controlling BVD virus infections are continually evolving. Current control procedures are based on identification and elimination of persistently infected cattle, which are a primary source of virus for non-infected cattle, and immunisation with killed or modified live virus vaccines. Additional concerns for control are the possible contamination of semen, embryos and biologicals with virus. In the near future, genetically engineered nucleic acid probes and subunit vaccines containing selected elements of the BVD virus may be available for incorporation into control procedures.     

Epidemiology of bovine virus diarrhoea virus

A better understanding of the epidemiology and pathogenesis of bovine virus diarrhoea virus (BVDV) has emerged in recent years. Fetal infections and in particular those resulting in birth of persistently infected calves are of central importance for the epidemiology of BVDV. A prevalence of persistently infected, viraemic animals of about 1% is found in Denmark and elsewhere by examination of randomly collected blood samples. A recent field study shows that 53% of randomly selected herds in an area in Denmark where BVDV is endemic had one or more persistently infected animals. Persistently infected cows may breed and will always transmit the infection to the calf. Such familial occurrence of persistent infection seems to be a fairly common phenomenon. Persistently infected cattle are important sources of infection to other cattle. Transiently infected cattle following experimental exposure will usually not transmit the infection by contact but this may not always apply to cattle after natural infection. Knowledge of the occurrence and potential for spread of virus from persistently infected bulls is reviewed. Virus is excreted with semen of both persistently and transiently infected bulls and BVDV may be transmitted by use of infected semen for insemination. The potential for spread of the infection through embryo transfer should be avoided by the use of adequate testing and controls.     

Hendra virus and Nipah virus animal vaccines

Hendra virus (HeV) and Nipah virus (NiV) are zoonotic viruses that emerged in the mid to late 1990s causing disease outbreaks in livestock and people. HeV appeared in Queensland, Australia in 1994 causing a severe respiratory disease in horses along with a human case fatality. NiV emerged a few years later in Malaysia and Singapore in 1998–1999 causing a large outbreak of encephalitis with high mortality in people and also respiratory disease in pigs which served as amplifying hosts. The key pathological elements of HeV and NiV infection in several species of mammals, and also in people, are a severe systemic and often fatal neurologic and/or respiratory disease. In people, both HeV and NiV are also capable of causing relapsed encephalitis following recovery from an acute infection. The known reservoir hosts of HeV and NiV are several species of pteropid fruit bats. Spillovers of HeV into horses continue to occur in Australia and NiV has caused outbreaks in people in Bangladesh and India nearly annually since 2001, making HeV and NiV important transboundary biological threats. NiV in particular possesses several features that underscore its potential as a pandemic threat, including its ability to infect humans directly from natural reservoirs or indirectly from other susceptible animals, along with a capacity of limited human-to-human transmission. Several HeV and NiV animal challenge models have been developed which have facilitated an understanding of pathogenesis and allowed for the successful development of both active and passive immunization countermeasures.     

Electron microscopy of bovine virus diarrhoea virus

Bovine virus diarrhoea virus (BVDV) has been (tentatively) identified by electron microscopy in purified virus preparations, in infected cell cultures and in tissues and cells from infected animals. These studies have revealed a spherical membrane-bound particle with a diameter of 40-60 nm. The membrane is smooth, bilaminar and surrounds a dense or semi-dense core of 20-25 nm. The core particle may be isometric or hexagonal. In studies of the morphogenesis of BVDV in infected cell cultures, it was found that assembly and maturation of the viral particles occur via a condensation process within membrane-bound vesicular organelles, in which the virions subsequently accumulate. Release of the virus occurs when the cell finally lyses and/or via exocytosis. Thus, both with regard to morphogenesis and to morphology, BVDV bears close resemblance to the Flaviviridae.     

Molecular biology of bovine virus diarrhoea virus

Our understanding of the molecular biology of bovine virus diarrhoea virus (BVDV) has greatly increased over the past several years. The development of monoclonal antibodies (MAB's) has identified a key antigen of BVDV while at the same time providing evidence for considerable variation in this protein. MAB's, particularly those directed against the p80 protein, can be developed for use in diagnostic tests while others may be useful in molecular epidemiological studies of BVDV. The successful cloning of BVDV and hog cholera virus can provide nucleic acid probes for use in routine diagnostic testing, for use in pathogenesis studies and for the detection of BVDV contamination in biological materials. With the identification of the key antigens of BVDV and its molecular cloning, the future holds the promise of vectored vaccines which can provide the efficacy of modified-live vaccines with the safety of killed vaccines. However, much work still must be done to define the significance of the antigenic variation of BVDV as it relates to providing protection for the developing fetus.     

Bovine virus diarrhoea virus: an introduction

In view of the recently established genome organisation of pestiviruses, their classification as members of the togavirus family is no longer tenable. They should rather be provisionally considered as a new genus of the Flaviviridae, irrespective of differences in the nonstructural genes. Like other positive-stranded RNA viruses, pestiviruses are highly variable; apart from point mutations, recombinations are expected to contribute to their capricious behaviour. One trait of expected pathogenetic significance in infections with bovine virus diarrhoea virus is a change from the non-cytopathogenic to a cytopathogenic biotype. Cooperation of both variants in an animal to produce the severe disease picture known as mucosal disease is unique in virology; elucidation of this mechanism may shed light on the pathogenesis of other sporadic diseases with suspected viral origin.     

Clinical aspects of bovine virus diarrhoea virus infection

Bovine virus diarrhoea virus (BVDV) infection of cattle results in a wide range of clinical manifestations. This article reviews the clinical responses associated with BVDV and discusses these diseases in terms of acute infection in immunocompetent cattle, fetal infection, infection in cattle immunotolerant to and persistently infected with BVDV and finally mucosal disease.     

The pathogenesis of bovine virus diarrhoea virus infections

Bovine virus diarrhoea virus (BVDV) disease in cattle ranges from the transient acute infections, which may be inapparent or mild, to mucosal disease which is inevitably fatal. On occasions the acute infections can lead to clinical episodes of diarrhoea and agalactia but as these syndromes cannot be reproduced experimentally, the pathogenesis remains unclear. The immunosuppressive effect of acute BVDV infections can enhance the clinical disease of other pathogens and this may be an important part of the calf respiratory disease complex. Although BVDV antigen has been demonstrated within the lymphoid tissues, for prolonged periods, the evidence for viral latency remains to be proven. Venereal infection is shown to be important in the transfer of virus to the foetus and congenital infections can cause abortions, malformations and the development of persistently viraemic calves. The two biotypes of the virus, non-cytopathogenic and cytopathogenic, are described. Their sequential role in the pathogenesis of mucosal disease arises from the initial foetal infection with the non-cytopathogenic virus and the subsequent production of persistently viraemic calves. These calves may later develop mucosal disease as a result of superinfection with a "homologous" cytopathogenic virus. The possible origin of this biotype by mutation is discussed. Chronic disease is defined as a progressive wasting and usually diarrhoeic condition; it is suggested that this may develop following superinfection of persistently viraemic cattle with a "heterologous" cytopathogenic biotype.     

Immunological responses to bovine virus diarrhoea virus infections

Infection of normal calves with bovine virus diarrhoea virus (BVDV) is a transient self-limiting infection that can result in a period of immunosuppression. The virus appears to be able to replicate in all of the major lymphocyte sub-populations as well as in accessory cells. This may result in the leukopenia that is often a sequel of infection and affects B-cells as well as the T-cell sub-populations expressing either BoCD4 or BoCD8 antigens. B and T-cell responses are affected as a consequence of exposure to BVDV and there is a reduced ability to control other infections. Evidence is summarised and shows that immunoglobulin is an important mediator of immunity to infection with BVDV. Although the foetus can mount an immune response in the latter part of gestation, during the first trimester it does not. A specific state of tolerance is induced and this is associated with change in the proportion of certain lymphocyte sub-populations and ability to respond to immune stimulation.