甲型肝炎病毒Vero细胞适应株的选育研究

将甲肝患者粪便中分离的甲型肝炎病毒在Vero细胞中进行适应性培养 ,选育高滴度适应株应用于甲肝灭活疫苗研究。在Vero细胞上连续传代 ,测抗原滴度和感染性滴度 ,满意后按WHO推荐的甲型肝炎灭活疫苗规程进行灭活疫苗试制研究。经Vero细胞 14次适应性传代后 ,病毒抗原滴度可高达 1∶2 5 6 0 ,感染性滴度为 8.2 3LogC CID50 /ml。试制的灭活疫苗HPSEC检测在 2 80nm时仅有一个高峰 ,SDS PAGE电泳 ,在 2 2kD、2 6kD和 33kD处有三条蛋白带 ,和HAVVP3、VP2和VP1的位置相同。ICR小鼠效力试验表明疫苗剂量 16 0 0EU/ml与Merck疫苗5 0U效果相似。通过研究获得了Vero细胞甲肝病毒适应株YN5株 ,初步证明可作为甲型肝炎灭活疫苗的候选毒株。     

呼吸道合胞病毒Long株的培养及应用

建立可稳定收获高滴度RSVLong株病毒的培养方法 ,所获得的RSVLong株病毒液用于RSV抗体ELISA检测。RSVLong株病毒 33℃传至 4代后 ,病毒滴度升高 ,出现典型的细胞病变。以RSVLong株病毒液作抗原所建立的ELISA试剂盒能检测出RSV特异性抗体。     

甲型肝炎病毒H2快速复制株在人二倍体细胞中的增殖研究

研究甲型肝炎病毒H₂快速复制株在人二倍体细胞中的生长特性,并缩短甲肝病毒的培养周期。将甲型肝炎病毒H₂株感染人二倍体细胞（KMB17细胞株）,采用高病毒感染复数（MOI）将病毒培养时间从26d缩短至10d后收获病毒,并通过连续传代进行适应研究,建立H₂株快速复制毒种库,在不同培养时间检测病毒抗原、感染性滴度,绘制病毒生长曲线,进行传代稳定性验证和病毒形态学的观察。甲肝H₂株快速复制病毒株在KMB17细胞上培养10d后收获,连续传代从第5代至第9代,抗原含量均在512～2 048之间,感染性滴度均在8.33 lgCCID₅₀/mL±0.125lgCCID₅₀/mL,H₂株快速繁殖至5代病毒和9代病毒在电镜下观察到多为成熟的实心颗粒。在5批次的重复试验中,病毒培养至10d、16d、22d时,收获的病毒感染性滴度无显著差异（P>0.005）。筛选后的甲型肝炎病毒H₂株的快速复制株缩短了甲肝病毒的培养时间,且保持较...     

ELISA法检测H3N2亚型流感病毒Vero细胞适应株

目的:流感病毒Vero细胞适应株A/Yunnan/1/2005va(H3N2)是一株以Vero细胞为培养基质能高产的病毒株,可用于制备以Vero细胞为基质的流感病毒裂解灭活疫苗;通过在低温下连续传代培养,可选育出流感病毒Vero细胞冷适应株,用于制备Vero细胞流感病毒减毒活疫苗为了便于对Vero细胞冷适应株的进一步研究,建立一个检测该病毒株的ELISA方法.方法:以流感病毒Vero细胞适应株A/Yunnan/1/2005Va (H3N2)的纯化抗原为免疫原制备羊抗A/Yunnan/1/2005Va (H3N2)和鸡抗A/Yunnan/1/2005Va (H3N2)的抗血清.将抗血清先后经硫酸铵沉淀法和Protein G亲和层析柱纯化后,以纯化的羊抗A/Yunnan/1/2005Va(H3N2) IgG为包被抗体,鸡抗A/Yunnan/1/2005Va(H3N2) IgG为第二抗体,测定包被抗体、第二抗体和酶标抗体最佳工作浓度,建立双抗体夹心ELISA检测方法.结果:羊抗A/Yunnan/1/2005Va (H3N2) IgG的最佳工作浓度为5μg/mL,鸡抗A/Yunnan/1/2005Va(H3N2) IgG的最适浓度为10 μg/mL,酶标抗体最佳稀释倍数为1∶4000.对已知的阳性样品,经双抗体夹心ELISA法测定的病毒滴度比血凝方法测定病毒滴度灵敏度高.结论:通过测定包被抗体、第二抗体和酶标抗体最佳工作浓度,建立了双抗体夹心ELISA检测流感病毒Vero细胞适应株病毒含量的方法.该方法操作简单、方便快速、敏感性高,可应用于Vero细胞冷适应株选育时对流感病毒Vero细胞适应株A/Yunnan/1/2005va(H3N2)的检测,对于研制Vero细胞流感减毒活疫苗有重要意义.     

肾综合征出血热双价纯化疫苗（Vero细胞）病毒株的筛选及其免疫原性研究

选用在Vero细胞上初步适应的LR1（Ⅰ型）、R22（Ⅱ型）株病毒,经乳鼠脑或腹腔接种,收剖感染鼠脑并在Vero细胞传代适应,此收获病毒与直接以Vero细胞传代的病毒比较表明：抗原效价、毒力滴度有明显提高。用LR1、LR22株病毒感染的Vero细胞培养物制备单价粗制疫苗和双价精制纯化疫苗免疫家兔,经空斑减少中和试验检测中和抗体,结果表明经乳鼠和Vero细胞交替传代适应后的LR1、R22株病毒具有良好的抗原性和免疫原性。     

用人胚肺二倍体细胞(2BS和SL7)分离甲型肝炎病毒

用我国建株的两株人胚肺二倍体细胞(2BS和SL7),以35℃为培养温度,直接从急性期甲型肝炎(简称甲肝)患者粪便中分离到4株病毒。该病毒在细胞培养上符合甲肝病毒的增殖特性,经免疫荧光阻断、免疫电镜及中和试验等特异性鉴定,证实为甲肝病毒。原始分离时的比较研究还显示,2BS株人二倍体细胞支持甲肝病毒增殖的能力似较SL7细胞佳。该4株病毒现已在2BS细胞稳定传代,感染滴度较高,被用于减毒活疫苗的育种研究。     

含甲肝病毒基因的重组痘苗病毒在动物体内产生针对甲肝病毒的中和抗体

用DNA重组技术得到的含甲肝病毒基因的重组痘苗病毒,可在家兔体内产生ELISA竞争抑制与中和抗体。基础免疫后,动物体内竞争抑制抗体滴度为10,加强免疫后达到80。由重组病毒产生的抗体中和指数比甲肝病毒产生者略低。     

蚕豆萎蔫病毒单克隆抗体制备及检测应用

:用蚕豆萎蔫病毒(BBWV)免疫的BALB/C鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,经筛选克隆,获得6株能稳定传代并分泌抗BBWV单克隆抗体(Mab)的杂交瘤细胞株,单抗腹水ELISA滴度为1:320000～1:640000,各单抗抗体类型均为IgG1。6株单抗与BBWV不同分离物均有反应,而与其它植物病毒无交叉反应。经Westernblot印迹分析表明,此6株单克隆抗体均是针对BBWV447kD的外壳蛋白大亚基的特异性抗体。这是国内外首次报道获得BBWV单克隆抗体     

狂犬病毒aG株在Vero细胞上传代适应研究

为研制有效、安全和稳定的Vero细胞狂犬病疫苗提供实验室基础资料。采用狂犬病固定毒4aG株在Vero细胞上进行传代适应,同时对该毒株在Vero细胞上的增殖条件,病毒液的回收方法进行研究。结果显示,狂犬病固定毒4aG株在Vero细胞上多次传代后获得一株Vero细胞适应株(4aG-V株),该毒株的毒力可达8.50 logLD50/ml,且具有很好的抗原性及免疫原性。结果表明,感染Vero细胞最适种毒比例为1∶103,病毒滴度随着时间的延长而增强到第12天后逐渐减弱,且采用低温冻融破碎法回收的病毒液滴度优于直接收液法。4aG-V株在Vero细胞上维持时间长,可连续收液,有望其生产高滴度的狂犬病毒液。     

Ⅱ型登革病毒D01090株在人胚肺二倍体细胞KMB17上适应株选育及初步鉴定

目的:选育能在人胚肺二倍体细胞KMB17上稳定增殖的Ⅱ型登革病毒适应株,为研发以人源性细胞为基质的登革疫苗候选株奠定基础。方法:将Ⅱ型登革病毒中国株D01090提取病毒基因组,通过RT-PCR法进行登革病毒型别鉴定后,在C6/36和Vero细胞上进行毒种扩增和滴度测定;将D01090毒株以4.0MOI接种KMB17细胞并反复传代至病毒完全适应在细胞内扩增,并连续传代10代,选育出良好的KMB17细胞适应株;病毒培养液经蔗糖梯度离心和超速离心后获得高浓度病毒液,接种KMB17细胞后通过透射电镜超薄切片检测细胞的病理变化;然后经过三轮蚀斑纯化筛选出纯化病毒株,免疫荧光法检测病毒纯化株的抗原性。结果:以Ⅱ型登革病毒中国株D01090基因组为模板,能扩增出511bp的登革病毒特异基因和119bp的Ⅱ型登革病毒型特异性基因。病毒经C6/36细胞扩增后滴度达4.5CCID50/ml,感染KMB17细胞至第三代可产生明显的细胞病变(cytopathic effect,CPE),连续传10代细胞病变速度逐渐增快,至第10代达到增殖高峰,病毒滴度达5.0CCID50/ml;将病毒感染6天后病变达+++的KMB17细胞进行超薄切片后经透射电镜观察细胞的病理变化,镜下可观察到内质网中新组装成的病毒颗粒,细胞周围产生很多分裂的小碎片,伴有游离出胞的病毒;三轮蚀斑纯化后筛选出纯化克隆,免疫荧光法检测病毒的抗原性呈阳性。结论选育出了能稳定传代且病毒扩增量高的Ⅱ型登革病毒KMB17细胞适应株,经蚀斑纯化后仍保持较好的抗原性。     

细胞培养结合实时荧光RT-PCR法快速检测甲肝病毒滴度的研究

目的建立一种细胞培养与实时荧光RT-PCR相结合的快速检测甲肝病毒滴度的方法。方法根据甲肝病毒(HAV)L-A-1株5'端基因组序列,设计了2条基因特异性引物及一条探针,建立实时荧光RT-PCR法,结合细胞培养检测甲肝病毒滴度,并与ELISA检测法进行比较。结果实验中建立的方法能特异检测甲肝病毒,细胞培养8d检测病毒滴度为lg107.0CCID50/mL。同一样本重复检测3次,批内样本Ct值的变异系数最大为0.89%,批间样本Ct值变异系数最大为1.66%。建立的细胞培养结合实时荧光RT-PCR法(细胞培养8 d)与细胞培养ELISA法(细胞培养28 d)检测甲肝病毒滴度结果差异无统计学意义(P0.05)。结论该方法具有快速、灵敏、特异等优点,应用于疫苗常规检测有良好前景。     

Dynamics of the accumulation of virus particles with different physico-chemical properties in FRhK-4 cells infected with hepatitis A virus

The propagation time-course of hepatitis A virus (HAV, strain HAS-15) in continuous culture of the foetal rhesus monkey kidney cells (FRhK-4) was investigated. The HAV infectivity and viral RNA content in the infected cells reached the maximal level 5-8 days after infection, while accumulation of hepatitis A antigen (HAAg) continued for 2-3 weeks more. Viral particles with the densities 1.27-1.28 g/cm3 and 1.18-1.22 g/cm3 were isolated from the infected cells as well as the mature virions with the buoyant density 1.33-1.34 g/cm3 in CsCl. The concurrent accumulation of mature virus and "light" particles (1.18-1.22 g/cm3) was registered during infection. Viral particles with the density 1.27-1.28 g/cm3 accumulated predominantly from the 14th to the 21st-24th days after infection. The mature virions (1.34 g/cm3) as well as the particles with the density 1.24-1.25 g/cm3 were isolated from supernatant precipitated by ammonium sulphate. The HAAg activity of both fractions increased progressively in equal proportion in course of infection.     

从抗体阳性的甲型肝炎接触者粪便中分离出一株甲型肝炎病毒

几年来,国内外已相继用组织培养法分离多株甲型肝炎病毒(HAV),并已证实了甲型肝炎患者及亚临床型感染在传染本病中的重要性。但HAV在流行点的正常人群中存在短期携带的问题还尚未见报道。本文采用人胚肺二倍体细胞(2BS)从甲型肝炎接触者粪便中分离出一株HAV,并经免疫电镜、免疫荧光及参比血清鉴定等证实。现将结果报告如下。     

甲肝病毒（龙甲—25株）在Frhk4细胞上传代特征

应用Frhk4细胞增殖HAV,经较系统研究,建立了稳定的细胞传代方法,病毒增殖条件,获得了较大量的甲肝病毒抗原.HAV龙甲-25株在2BS细胞上传至第六代移到Frh4细胞上连续传至九代,用荧光法(IF)、EMISA夹心法,和免疫电镜方法(IEM)进行检测,结果表明龙甲-25不同代次间的病毒增殖情况不同.我们将龙甲-25HAV在Frhk4细胞上从第七代传至第十六代,分别用IF,ELISA,IEM方法检测,结果是细胞在感染病毒后不同天数的IF结果阳性,病毒传代的代次不同在ELISA和IEM检测中出现不同的结果.在第九代至第十一代的HAV′培养过程中我们改变了病毒培养及收获方法,再重复试验时,出现三种实验方法一致的结果.     

Effect of hepatitis A virus infection on cell metabolism in vitro

Hepatitis A virus (HAV), when inoculated into cultures of the PLC/PRF/5 cell line which produces the surface antigen of hepatitis B virus (HBsAg), showed growth characteristics different from those of other picornaviruses. Antigen of HAV (HAAg) is expressed only about 10 days after infection. No major impact on the overall macromolecular biosynthesis of the host cells is observed. The growth rate of HAV-infected and uninfected cells was comparable, although the plating efficiency of infected cells was lower. Different hormonal factors were tested for their ability to stimulate viral antigen expression. Dexamethasone or prostaglandin E1 added to the culture medium increased HAAg expression; insulin reduced expression. Persistent infection of hepatoma cells by HAV never led to a cytolytic infection. In temperature-shift experiments, an adverse effect on the expression of HAAg and HBsAg was observed. In all experiments, the amounts of HBsAg in HAV-infected cells were reduced. On the whole, no major influence on host-cell metabolism is observed in cells persistently infected with HAV. Cell-mediated immunological response as a mechanism of pathological changes in HAV-infected liver is, therefore, more likely than a cytopathological effect.     

Characterisation of Haemonchus contortus-derived cell populations propagated in vitro in a tissue culture environment and their potential to induce protective immunity in sheep

Cell populations derived from viable Haemonchus contortus L(3) larvae were propagated in vitro in a tissue culture environment for a prolonged period (>48 months). Microscopic evaluation of H. contortus-derived cell populations revealed gross morphological characteristics highly analogous to those described for cell types originating from species of plant nematodes propagated in vitro in a tissue culture environment for a briefer period of time (<6 months). The characterisation of extracts harvested from tissue culture populations of H. contortus-derived cells by SDS-PAGE analysis detected molecular fractions of approximately 29, 45, 55, and 200-kDa that closely correlated with reports for preparations obtained from intact/viable H. contortus larvae. Complementary investigations detected the dual biochemical expression of phosphohydrolase and aminopeptidase-M activities based on the hydrolysis of the synthetic enzyme-specific substrates, para-nitrophenylphosphate and leucine-para-nitroanaline, respectively. The identification of phosphohydrolase and aminopeptidase-M-like biochemical activity in fractions harvested from H. contortus-derived cell populations and propagated in vitro in tissue culture served as evidence validating their parasitic-origin. Further validation of H. contortus-derived cell populations propagated in tissue culture entailed the formulation of Triton X-100 extracts containing potential immunoprotective antigens with SEAM adjuvant and its administration by intramuscular injection (100 microg total protein) to healthy sheep (n=8) on day 0 (left rear-limb) and day +14 (right rear-limb). Animals on day 28 subsequently received a single oral challenge of 10,000 infective L(3)-stage H. contortus larvae. Applying ELISA methodologies, increases in antigen-specific IgM and IgG were detected in ovine serum samples. Interpretation of experimental findings revealed that sheep with the greatest antigen-specific humoral immune responses (IgG titre 1/3125) also demonstrated a degree of reduced abomasal H. contortuslarvae burdens (60% reduction). Polyclonal antibody from immunoprotected sheep was subsequently found to recognise both the: (i), digestive tract; and (ii), antigen extracts associated with intact/viable H. contortus larvae. These experimental findings reveal the potential feasibility of propagating parasite-derived cell populations in an in vitro tissue culture environment in a manner that retains their ability to express immunoprotective antigenic fractions.     

用生物素-亲和素ELISA法检测人胚肺二倍体细胞培养的甲型肝炎病毒抗原

利用生物素-亲和素生物放大系统的原理,建立了新的检测细胞培养的甲型肝炎病毒抗原的方法。用生物素标记的抗甲肝IgG,与免疫偶联于固相的甲肝抗原作用,然后用亲和素-生物素辣根酶复合物(ABPC)与生物素化的IgG偶合,邻苯二胺(OPD)显色。该法在检测甲肝抗原中获得满意结果,灵敏度比普通ELISA法提高约10倍,非特异性显著降低。该法可进一步发展成为一种高特异性的,灵敏的甲肝临床诊断和流行病学调查的技术。     

Localization of hepatitis A antigen in liver tissue by peroxidase-conjugated antibody method: light and electron microscopic studies.

Hepatitis A antigen (HAAG) was localized in liver tissue from marmosets inoculated with human hepatitis A virus (HAV) by light and electron microscopy by using a peroxidase-conjugated antibody method. The fine granular peroxidase staining was scattered throughout the cytoplasm of liver cells when viewed with the light microscope. The distribution of HAAg-positive cells was focal. Virus-like particles, 24 to 27 nm in diameter, were observed in the cytoplasm of hepatocytes and smaller cells, resembling Kupffer cells, by standard thin-section electron microscopy (thin section EM). By immunoperoxidase electron microscopy (immunoperoxidase EM), HAAg was detected on the particles, which were aggregated within cytoplasmic vesicles of the hepatocyte. The surrounding membrane of the vesicles was also HAAg- positive. Similar HAAg particles were observed in the cytoplasm of smaller cells adjacent to hepatocytes as well. Thus, immunoperoxidase EM revealed that the 24- to 27-nm virus-like particles in the cytoplasm of liver cells obtained from marmosets were infected with HAV contained HAAg.