共查询到20条相似文献，搜索用时 31 毫秒
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【目的】通过建立过表达T7 RNA聚合酶(T7 RNA polymerase, T7 RNAP)的慢病毒系统,构建可稳定表达T7 RNAP的BHK-21细胞株。【方法】根据GenBank中公布的大肠杆菌BL21(DE3)基因组中T7 RNAP基因序列(登录号：NZ＿CP081489.1)设计引物,并采用PCR扩增T7 RNAP基因片段,将其克隆至慢病毒载体中,构建pCDH-CMV-T7 RNAP重组质粒。筛选阳性克隆及测序鉴定后,利用脂质体将包含重组质粒pCDH-CMV-T7 RNAP的包装系统和辅助包装质粒共转染至HEK-293T细胞,进行慢病毒的包装及纯化,获得高病毒滴度的重组慢病毒。将收集的病毒液分别以感染复数(multiplicity of infection, MOI)为1、5、10和20感染BHK-21细胞,72 h后观察荧光情况,筛选最佳MOI。将重组慢病毒在最佳MOI条件下感染BHK-21细胞,通过绿色荧光蛋白copGFP的表达观察感染细胞的阳性率。使用4μg/mL嘌呤霉素作为抗性筛选物进行多轮筛选,最终筛选得到BHK-21-T7 RNAP细胞株。进一步对所筛选的细胞株... 相似文献
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M Onuma S Watarai M Sonoda T Mikami H Izawa 《Canadian journal of veterinary research》1980,44(3):289-293
When various indicator cells, including virus transformed and nontransformed cells, were cocultivated with bovine leukemia virus-producing cells, strong positive syncytia formation was found in transformed cells one day after cocultivation. The results of comparison of bovine leukemia virus antibody titers and the detection of bovine leukemia by the syncytium assay showed 89% of serologically positive cows were positive for bovine leukemia virus, whereas no reactors were found in serologically negative cows. However, the frequency of bovine leukemia virus detection differed according to the difference of incubation periods in the syncytium assay. Therefore, it is important to choose the appropriate indicator cell and culture conditions for the detection of bovine leukemia virus in the syncytium assay. 相似文献
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Comparative studies on the growth of Australian bluetongue virus serotypes in continuous cell lines and embryonated chicken eggs 总被引：2，自引：0，他引：2
The replication of cell culture passaged Australian bluetongue virus (BTV) isolates, serotypes 20 (CSIRO19) and 1 (CSIRO156), and an untyped BTV (CSIRO154) was assessed in eight continuous cell lines (one derived from baby hamster kidney cells, BHK-21; three derived from monkey kidney cells, Vero, LLC-MK2 and CV-1P; a foetal ovine lung and a mouse fibroblast cell line, CSL503 and L929, respectively, a Super-Vero-Porcine stable cell line, SVP; and a mosquito cell line, Aedes albopictus cells) and in 11-day-old embryonated chicken eggs (ECE) at different multiplicities of infection. All three viruses replicated in the cell lines tested, maximum extracellular virus yields being attained from BHK-21 cells at high multiplicities of infection (approximately 10 PFU per cell). Also BHK-21 cells produced much higher yields of virus than the other cell lines tested when low multiplicities of infection were used (approximately 10(-4) PFU per cell). All BTV serotypes multiplied in Singh's Aedes albopictus cells with no cytopathogenic effects over the 4 day period tested. The viruses also replicated in 11-day-old ECE; however, the sensitivity of ECE for growth of the Australian serotypes was not as high as has been reported for BTV isolates in other countries. In all cell culture systems and in ECE, BTV1 and BTV20 replicated more efficiently than did CSIRO154 virus. 相似文献
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采用HE、Giemsa染色、透射电镜以及DNA琼脂糖凝胶电泳等研究了水泡性口炎病毒（VSV）诱导BHK-21细胞凋亡的过程。结果显示：VSV感染BHK-21细胞后,光镜下可见细胞圆缩,细胞器固缩、核仁消失、染色质凝聚和核碎裂、凋亡小体出现;电镜下观察到染色质聚集形成典型的新月形,胞浆中充满大量空泡,细胞核因染色质凝聚也发生了空泡化;1％的琼脂糖凝胶电泳出现180-200bp整倍数的DNA梯形条带。结果表明,VSV诱导BHK-21细胞凋亡是其致细胞病变的主要表现形式之一。 相似文献
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试验利用口蹄疫病毒感染BHK-21细胞,通过MTT法、Hoechst 33258染色、原位末端标记技术(TUNEL)、流式细胞术和链特异性荧光定量RT-PCR,分别就口蹄疫病毒对BHK-21细胞生长的抑制作用、凋亡细胞的形态学和分子生物学特征、凋亡峰的出现和细胞周期的变化以及口蹄疫病毒基因组在BHK-21细胞内的复制情况进行了检测。结果表明:口蹄疫病毒可抑制BHK-21细胞的生长并诱导其产生凋亡,呈现典型的凋亡细胞特征,出现细胞凋亡峰并且细胞周期明显被阻滞在G1/G0期,同时对凋亡率和口蹄疫病毒基因组复制的关系做了初步研究。 相似文献
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金丝桃素体外抗口蹄疫病毒与宿主细胞吸附作用研究 总被引：3，自引：0，他引：3
对光活化金丝桃素体外抑制口蹄疫病毒与宿主细胞吸附作用进行实验。体外培养的BHK-21细胞加入金丝桃素,日光灯下光活化1h后感染FMDV。通过细胞病变观察、MTT法、双抗体捕获抗原法、扫描电子显微镜进行抑制FMDV与宿主细胞吸附、融合研究。结果表明金丝桃素有明显抑制细胞病变的作用。双抗体捕获抗原法检测金丝桃素有效抑制FMDV和BHK-21细胞的吸附、融合,抑制率可达59．72％。扫描电镜观察,金丝桃素组与病毒对照组相比,BHK-21细胞FMDV颗粒明显减少。提示金丝桃素在体外有显著抑制口蹄疫病毒与宿主细胞的吸附、融合作用。 相似文献
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S J Wechsler K J Austin W C Wilson 《Journal of veterinary diagnostic investigation》1990,2(2):103-106
The sensitivity of different assay systems for detecting low concentrations of bluetongue virus (BTV) were compared. These assays included blind passage on baby hamster kidney (BHK-21) cells and on cattle pulmonary artery endothelial (CPAE) cells, immunoperoxidase staining of cells on multiwell slides, and cDNA/RNA hybridization of BTV infected cells. Nine serial 10-fold dilutions of a cell culture-adapted BTV serotype 11 were tested (each dilution was treated as a separate sample) in all assays. Visual inspection for cytopathic effects (CPE) during 3 passages in BHK-21 cells detected samples that contained greater than or equal to 3 plaque forming units (PFU)/ml of BTV. Evidence of CPE during 3 passages in CPAE cells detected samples that contained greater than or equal to 0.3 PFU/ml of BTV. A limit of detection (greater than or equal to 0.3 PFU/ml) was obtained faster by immunoperoxidase staining of BTV-inoculated CPAE cells on multiwell slides and incubated for 3 days. The cDNA/RNA hybridizations of CPAE and BHK-21 cells incubated for 2 or 3 days, respectively, with BTV dilution samples detected samples that contained greater than or equal to 30 PFU/ml. Of the assay systems examined, immunoperoxidase staining of CPAE cells on multiwell slides inoculated with cell culture-adapted BTV was the most sensitive and fastest assay for definitive virus identification. 相似文献
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本研究旨在对鸡新城疫病毒(Newcastle disease virus,NDV)LaSota株在BHK-21细胞上的增殖条件进行优化,为实现应用细胞系生产NDV提供参考。在确定BHK-21细胞TPCK-胰酶耐受浓度的基础上,在培养基中添加不同浓度的TPCK-胰酶、新生牛血清及调节不同pH,通过对细胞培养液细胞半数感染量(TCID50)的测定,确定最适胰酶浓度、血清浓度及pH;通过固定接毒量而添加不同数量细胞或固定细胞数量而接种不同量的病毒,确定最适细胞量及接毒量;将BHK-21细胞接种NDV LaSota株后,通过测定不同时间细胞上清液TCID50绘制增殖曲线,确定最佳收毒时间;应用转瓶对该病毒进行扩大培养,并进行TCID50、鸡胚半数感染量(EID50)与血清凝集价(HA)的测定。结果显示,NDV LaSota株在BHK-21细胞上增殖的最适TPCK-胰酶浓度为3μg/mL,最适接毒量为106.375 EID50,细胞数量为1×10~6个,培养基pH为7.2,且可用无血清培养基进行培养,BHK-21细胞接种NDV LaSota株后最佳收毒时间为34～36 h。应用BHK-21细胞采用转瓶培养的方式对NDV LaSota株进行增殖,结果显示,细胞培养液TCID50值为107.0/0.1 mL,EID50为107.5/0.1 mL,HA效价为1∶256。因此,应用以上条件增殖培养NDV LaSota株,完全适用于该病毒的规模化生产,且符合现行的兽医生物制品与检验制造规程。 相似文献
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伪狂犬病病毒 (Ｐｓｅｕｄｏｒａｂｉｅｓｖｉｒｕｓ ,ＰｒＶ)属疱疹病毒科α 疱疹病毒亚科 ,能引起多种家畜及野生动物的伪狂犬病 ,尤其是猪的伪狂犬病 ,已成为危害当今养猪业的最严重的传染病之一。根据已成功根除伪狂犬病国家的经验以及伪狂犬病病毒分子生物学研究的新成果 ,种猪的免疫还是以灭活苗为主。但传统的灭活苗由于缺少检测标志 ,无法采用鉴别诊断方法区分疫苗免疫猪和野毒感染猪。而在目前广泛使用的三种基因缺失标志疫苗株 (ｇＧ- 、ｇＥ- 、ｇＣ- )中 ,由于 ｇＧ的缺失不影响免疫原性 ,因此 ,ｇＧ- 灭活苗优于 ｇＥ- 、ｇＣ-… 相似文献
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旨在筛选伪狂犬病病毒（PRV）敏感的BHK-21细胞并分析其生长和病毒增殖特性,优化反应器中BHK-21悬浮细胞的培养和病毒增殖条件,建立生物反应器培养BHK-21悬浮细胞增殖PRV工艺。本研究利用响应面和单因素优化法,以细胞生长动力学特性、TCID50病毒滴度等参数为指标,优化1.2 L生物反应器中BHK-21悬浮细胞的最佳培养和增殖病毒条件,在5 L生物反应器中进一步批培养验证。结果显示,筛选获得PRV高敏感的BHK-21-02贴壁细胞和BHK-21-XF02悬浮细胞各1株,BHK-21-XF02悬浮细胞在含3%血清的SLM-BHK低血清培养基和SFM-BHK无血清培养基中均能实现良好的生长和病毒增殖。利用响应面法优化得到1.2 L反应器最佳培养条件为接种密度1.20×106cells·mL-1、搅拌转速120 r·min-1、DO值40%,5 L反应器批培养72 h细胞密度可达（7.61±0.18）×106 cells·mL-1、细胞活率为（96.93±1.18）%。利用单因素法优化得到1.2 L反应器最佳病毒增殖条件为MOI 0.001、培养温度37℃、细胞密度2.0×106cells·mL-1、搅拌转速80 r·min-1,5 L反应器批培养接毒后48 h病毒滴度达到最大值（7.13±0.11） lgTCID50·mL-1。本研究可为PRV疫苗相关研究和规模化生产提供参考。 相似文献
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Induction of antibodies in mice by a recombinant baculovirus expressing pseudorabies virus glycoprotein B in mammalian cells. 总被引：5，自引：0，他引：5
The glycoprotein gB of pseudorabies virus (PrV) was expressed in various mammalian cells by a recombinant baculovirus carrying the PrV gB gene under the control of the CAG promoter. When the recombinant baculovirus was inoculated into the stable porcine kidney cell line CPK, expression of PrV gB was detected by immunofluorescent antibody analysis and a 155 kDa of protein, which has the same molecular mass as the native PrV gB, was detected by Western blotting. High levels of expression of PrV gB were observed in BHK-21, HmLu-1 and SK-H cell lines. Furthermore, anti-PrV gB-specific antibodies against PrV gB protein were detected by the enzyme-linked immunosorbent assay in mice inoculated the recombinant baculovirus. The recombinant baculovirus containing the PrV glycoprotein gB gene under the CAG promoter could be a candidate for a pseudorabies vaccine. 相似文献
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伪狂犬病病毒吉林分离株感染BHK-21细胞的超微结构变化 总被引：1，自引：0，他引：1
以猪伪狂犬病病毒(PRV)吉林分离株PRV-JL感染体外培养的BHK-21细胞为模型,通过透射电镜对PRV的形态发生学和宿主细胞超微结构的动态变化规律进行研究。结果显示,PRV能导致BHK-21细胞圆缩,并发生细胞融合,形成合胞体;电镜观察到的病毒粒子呈球形或椭圆形,成熟的病毒粒子直径大小为140~210 nm,未成熟病毒粒子直径为90~150 nm,多呈中空状,部分呈致密核芯。病毒吸附于细胞后以膜融合的方式进入细胞,在胞核内复制,装配好的病毒粒子以出芽的方式离开细胞核,获得最初的囊膜,进入胞浆;在胞浆内的病毒粒子又利用高尔基体的膜结构合成第2层囊膜,形成完整的病毒粒子;最后包裹有完整病毒粒子的高尔基囊泡与细胞膜发生融合,将病毒粒子释放到细胞外。感染细胞超微结构变化主要表现为:细胞胞浆空泡增多,内质网扩张,线粒体增生、嵴肿胀、脱落,最后空泡化,整个细胞裂解、破碎。 相似文献
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Y Tsukamoto T Kotani K Kohama S Sakuma F Sasaki 《Canadian journal of veterinary research》1999,63(4):282-283
Cultured chick embryonic fibroblasts formed syncytia after infection with avian reovirus (ARV) strain 58-132. Mitotic figures were occasionally observed within the syncytia. In addition, many nuclei in the syncytia incorporated 5-bromo-2'-deoxyuridine (BrdU), a DNA replication marker, indicating that they were in the S-phase of the cell cycle. These observations suggested that the nuclei within ARV-induced syncytia originated from nuclear endomitosis without cell division, as well as from cell fusion. 相似文献
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Lan NT Yamaguchi R Kai K Uchida K Kato A Tateyama S 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2005,67(5):491-495
To know growth profiles of canine distemper virus (CDV) on Vero cells stably expressing canine signaling lymphocyte activation molecule (Vero-DogSLAMtag; Vero-DST cells), the propagation of three strains of CDV was tested in Vero-DST cells in comparison with parental Vero cells. Strain MD77 could grow well in both cell lines, but demonstrated no syncytium formation or indistinguishable rounding cytopathic effects (CPE) in Vero cells. Strains Onderstepoort and KDK-1 also grew well in Vero-DST cells with apparent syncytium CPE, while they grew less or no efficiently, respectively, in Vero cells. All three CDV strains demonstrated the peak titers, in Vero-DST cells before reaching to an extensive CPE and drastic decrease of titers at/after full CPE. Immunohistochemistry revealed that viral antigens of all CDV strains were found exclusively in the syncytia in Vero-DST cells, while in Vero cells, viral antigen was identified in their single cells for strain MD77 but none for other strains. Thus, every strain of CDV could grow well in Vero-DST cells and behaved differently against Vero cells. These results would be of practical value for workers of CDV because 1) In Vero-DST cells, by observation of distinct syncytium CPE, the highest titer or the best growth of virus could be identified; 2) In Vero cells, various CDV strains could be readily classified after propagation in Vero-DST cells. 相似文献
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从送检的2日龄病猪脑部分离到1株病毒。该病毒接种的Balb／c小鼠出现了典型的伪狂犬病症状,接种BHK-21细胞48h后出现了圆缩、集聚,脱落等典型的细胞病变,猪狂犬病毒阳性血清能特异性的中和该分离病毒。根据GenBank公布的PRV的gE基因设计的引物能扩增出特异性片段,证实该病毒为伪狂犬病毒。 相似文献
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Cell cultures infected with BHV-1/F(syn), a recombinant bovine herpesvirus 1 (BHV-1) which expresses a synthetic open reading frame encoding the fusion (F) protein of the bovine respiratory syncytial virus (BRSV), showed a cytopathic effect (CPE) indistinguishable from that induced by wildtype BHV-1 although transient transfection experiments demonstrated that expression of the F protein leads to formation of large syncytia. Since it has been shown that glycoprotein M (gM) of pseudorabies virus inhibits BRSV F-induced syncytium formation in transient plasmid transfection experiments [Pseudorbies virus glycoprotein M inhibits membrane fusion. J. Virol. 74 (2000) 6760], the gM ORF of wtBHV-1 and BHV-1/F(syn) was interrupted. Infection of cell cultures with the resulting gM(-) mutant of BHV-1/F(syn) led to formation of syncytia, whereas the CPE in gM(-)BHV-1 infected cells was comparable to the CPE in wtBHV-1 infected cultures. Our results demonstrate that gM is not essential for BHV-1 replication in cell culture and that gM is involved in inhibition of the cell fusion activity of the BHV-1 expressed BRSV F protein. 相似文献
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试验旨在构建能高效表达非洲猪瘟病毒（African swine fever virus,ASFV） p54蛋白的重组伪狂犬病病毒（Pseudorabies virus,PRV）。通过无缝克隆构建含有PRV TK基因同源臂和绿色荧光蛋白（GFP）报告基因的PRV通用转移载体pCAGIG-TK（l+r）,参考China/2018/AnhuiXCGQ株基因序列,优化合成E183L基因,并连入通用转移载体,构建重组中间转移质粒pCAGIG-TK（l+r）-p54;重组质粒ScaⅠ酶切线性化后经脂质体介导转染BHK-21细胞,6 h后感染0.1个感染复数（MOI） PRV变异株,出现病变后通过空斑挑选和PCR鉴定纯化重组PRV,进一步通过Western blotting和间接免疫荧光试验（IFA）检测外源蛋白表达,并对重组病毒的遗传稳定性和增殖特性进行研究。结果显示,转染重组质粒pCAGIG-TK（l+r）-p54的BHK-21细胞24 h后可观察到绿色荧光,说明重组质粒成功转入BHK-21细胞;纯化后的重组病毒表达绿色荧光蛋白且含有ASFV E183L基因,Western blotting和IFA结果证实重组PRV在BHK-21细胞中能表达p54外源蛋白,在26 ku处呈现特异性条带,且能与ASFV阳性血清发生特异性反应,免疫原性较好;重组病毒在BHK-21细胞上连续传代20次均能检测到ASFV E183L基因,遗传稳定性较好;一步生长曲线显示,重组病毒与亲本病毒的增殖特性差异不大,且二者的最高病毒滴度分别为107.34/0.1 mL和107.61/0.1 mL,外源片段的插入不影响重组病毒在细胞上的增殖能力。本研究成功获得了表达ASFV p54蛋白的重组病毒rPRV-p54,为进一步研究p54蛋白的免疫原性及开发非洲猪瘟多基因重组PRV载体疫苗奠定了基础。 相似文献
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为建立基于无血清悬浮培养细胞生产新城疫病毒(NDV)的工艺,本研究首先筛选了适于NDV增殖的乳仓鼠肾细胞(BHK-21)单克隆细胞株,并将鸡胚适应的NDV在筛选获得的细胞株(BHK-v002)中传代,获得细胞适应的NDV。进一步采用单因素实验法检测病毒感染复数(MOI)、TPCK-胰酶浓度、细胞培养液的稀释比例等工艺参数对病毒效价的影响。结果显示,NDV在无血清培养的BHK-v002细胞中增殖的最适条件为:当细胞生长至约9.0×10^6个/mL时,以培养液.新鲜培养基为2:1的比例补加新鲜培养基,使细胞密度达6.0×10^6个/m L,按MOI为0.005接种NDV LaSota株,TPCK-胰酶终浓度为5μg/mL。接种病毒后96 h收获病毒液的HA效价为8.5 log2HAU/25μL,单细胞产毒量(Svy)达到1 685.9病毒颗粒/细胞,半数组织细胞感染剂量(TCID50)为7.9 log10TCID50/100μL。本研究确定了NDV LaSota株在BHK-21细胞悬浮培养中的增殖条件,建立了基于BHK-21细胞无血清悬浮培养体系中NDV的生产工艺,该工艺操作简便,易于放大,为当前ND疫苗的鸡胚生产工艺提供了候选替代方案。 相似文献
20.
Safety and immunogenicity of ERA strain of rabies virus propagated in a BHK-21 cell line. 总被引：5，自引：2，他引：3 
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下载免费PDF全文 K F Lawson R Hertler K M Charlton J B Campbell A J Rhodes 《Canadian journal of veterinary research》1989,53(4):438-444
The ERA strain of rabies virus was propagated in a baby hamster kidney cell line (BHK-21/C13). The viral titer was 10(1.8) tissue culture infective doses (TCID) higher than that of commercial ERA vaccine. The ERA/BHK-21 vaccine in baits retained titers of 10(6.3) to 10(6.4), TCID when subjected to daily temperature fluctuations from 9 degrees C to 24 degrees C for 21 days. This titer, according to a dose response in laboratory foxes, was still capable of immunizing up to 100% of foxes consuming a bait. The ERA/BHK-21 vaccine, when presented in baits, produced antibodies in 80 to 100% of dogs consuming more than one bait. Duration of immunity in foxes, from feeding the ERA strain rabies virus in baits, as determined by resistance to challenge with virulent virus, was at least 48 months. The vaccine strain retained some pathogenicity for nontarget species. In tests carried out on foxes, raccoons, dogs, cats and cattle, the vaccine did not cause vaccine-induced rabies. One of 14 skunks which consumed four baits developed vaccine-induced rabies, but virus could not be isolated from the salivary glands of this animal. The vaccine, when presented in baits, caused vaccine-induced rabies in 37% of laboratory mice, 3.4% of Microtus and 2.6% of Peromyscus species. Rabies virus could not be isolated from the salivary glands of rodents with vaccine-induced rabies. It was concluded that ERA virus propagated in BHK-21/C13 cells and incorporated in an acceptable bait produced a high titer, stable, immunogenic and safe vaccine for foxes. 相似文献