狂犬病毒CTN—1株在Vero细胞上的适应传代研究

本文报导了用我国狂犬病毒固定毒人二倍体细胞适应株（ＣＴＮ－１）进行Ｖｅｒｏ细胞适应传代研究。通过连续传代培养,滴度可达８．０１ｏｇＬＤ５０／ｍｌ,达到了ＷＨＯ规定的不需浓缩的标准。病毒用０．０１ＭＯＩ感染细胞其产量与１Ｍｏｌ感染量相仿。病毒增殖高峰在４－５天,维持达１５天无明显下降,且可连续收获４－５次。因此,该毒种符合ＷＨＯ提出的疫苗生产毒种要求,可用于狂犬病疫苗生产。     

流行性乙型脑炎病毒在Vero细胞上传代适应的研究

通过乙脑病毒Ｐ＿３株以不同方式在鼠脑和Ｖｃｒｏ细胞间传代适应的研究,培育出适于工业化大生产制备乙脑疫苗的生产毒种。其毒力可达７．０±０．５ｌｇＬＤ５０／０．０４ｍｌ,空斑滴度为７．５±０．５ＰＦＵ／ｍｌ,免疫原性ＩＤ５０值为０．００００１６－０．００００２３ｍｌ;在含有适量人白蛋白的１９９营养液内,该毒种于－３０℃至少可稳定一年;与鼠脑组织毒种相比,传代细胞毒种的毒力、免疫原性及稳定性均与其相当。然而,后者不含脑组织,至少降低了一种过敏的危险。另外,细胞毒种的制备无需手工解剖鼠脑,易于控制外源因子污染,易于标准化。对于大规模生产疫苗、提高疫苗产量和质量,传代细胞毒种比鼠脑组织悬液毒种具有明显的优点。     

人用精制Vero细胞狂犬病疫苗的保护性试验

用ＣＴＮ－１Ｖ株生产的精制Ｖｅｒｏ细胞狂犬病疫苗腹腔免疫小鼠后,再用狂犬病毒街毒株ＳＢＤ０７脑内和肌肉攻击,结果表明稀释５倍疫苗的保护率分别为８８．９％和９０％,且５倍稀释疫苗的保护效果与原苗相当,此研究证明ＣＴＮ－１Ｖ株能用来作为疫苗的生产毒株。     

大规模区带离心纯化Vero细胞乙脑疫苗

本文报道一种适合疫苗生产的大规模纯化Ｖｅｒｏ细胞乙脑疫苗的方法。原疫苗经适当浓缩和去除ＤＮＡ处理后,用不连续蔗糖梯度（３６％和６０％）。３２６００ｇ,速率区带离心４ｈ。纯化后疫苗的效力比中国参考疫苗高出６倍以上,补体结合抗原比中国参考疫苗高４～８倍。总蛋白含量低于３０μｇ／ｍＬ,牛血清含量降至０．５μｇ／ｍＬ以下,细胞残余ＤＮＡ低于１００ｐｇ／０．５ｍＬ。用此法连续制备三批纯化疫苗,其纯度和效力均高于日本鼠脑纯化疫苗。此法对于制备其它纯化Ｖｅｒｏ细胞疫苗也具有一定的参考意义。     

传染性法氏囊病病毒弱毒株对Vero细胞的适应性研究

传染性法氏囊病病毒疫苗的传统生产工艺是利用鸡胚纤维细胞增殖ＩＢＤＶ,但易被外源病原污染,且生产成本很高,借鉴国内外流行性乙型脑炎疫苗生产的经验,使用Ｖｅｒｏ细胞增殖ＩＢＤＶ弱毒株,从病毒高峰出现时间、滴度峰值的高低、对细胞代谢的影响等方面研究了ＩＢＤＶ对Ｖｅｒｏ细胞的适应性,敏感性以及适应后的种毒的长期保存过程中发现病毒滴度有下降趋势,试用了不同试剂对病毒进行保护。     

应用Vero细胞检定法和家兔皮内中和法检测吸附白喉类毒素的效价

Ｖｅｒｏ细胞法测定ＤＴＰ疫苗中白喉类毒素的效价与家兔皮内中和法（简称ＮＴ法）所得结果两法之间有一种平行关系,并且有很好的相关性,ｒ＝０．９３。但Ｖｅｒｏ细胞法测得平均值低于ＮＴ法,二者之间的平均比率为０．２８７,Ｓ＝０．０８９。本试验结果与１９９５部颁规程ＮＴ法０．２５ＩＵ／ｍｌ相比,确定了Ｖｅｒｏ细胞法以０．０７ＩＵ／ｍｌ作为检定吸附ＤＴＰ效价的最低要求。     

肾综合征出血热病毒LR1株在Vero细胞上适应的特性研究

选取不同代次的ＬＲ１毒株在Ｖｅｒｏ细胞上传代适应,不同天数连续动态观察细胞病变情况,同时用免疫荧光法和ＥＬＩＳＡ进行抗原检测,收毒前细胞反复冻融及超声波破碎,细胞破碎前后用ＥＬＩＳＡ检测抗原含量,半微量空斑法检测病毒滴度。结果证明：ＬＲ１株病毒能在Ｖｅｒｏ细胞上产生细胞病变,病变程度与其毒力明显相关;ＬＲ１株病毒在Ｖｅｒｏ细胞上繁殖的最佳收毒时间为１１天左右;病毒释放性较差,冻融和超声波破碎细胞能明显提高其抗原含量和病毒滴度     

浓缩地鼠肾细胞狂犬病疫苗与纯化Vero细胞狂犬病疫苗接种 …

为了解国产浓缩地鼠肾细胞狂犬疫苗与法国纯化Ｖｅｒｏ细胞狂犬疫苗接种人体后中和抗体产生情况。分别用两种疫苗接种１０人,用小鼠中和试验方法检测中和抗体滴度。国产疫苗五针全程免疫后３０天（第一针按种后６０天）可１００％达到保护水平,法国疫苗在第一针接种后３０天１００％达到保护水平。第一针接种后１４、３０、６０天时,前者抗体平均滴度分别是０．０６ＩＵ／ｍｌ、１０２ＩＵ／ｍｌ、２．０７ＩＵ／ｍｌ;后者抗体平     

狂犬病病毒Vero细胞适应株3aG-V的致病性和免疫原性

狂犬病病毒3aG—V株是3aG—5在vero细胞适应的毒株,对其在小鼠、豚鼠、家兔、犬的不同感染途径的致病性,小鼠、豚鼠、兔子上的免疫原性进行研究,同时进行纯毒及在中枢神经系统中能否形成尼氏小体试验,结果表明：狂犬病病毒3aG—V株潜伏期较长,在动物脑内不形成尼氏小体,为固定毒,其在实验动物上具有较弱的致病性和较好的免疫原性,可作为纯化Vero细胞狂犬病疫苗的生产用疫苗株。     

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。     

流感病毒在Vero细胞上的增殖

目的研究流感病毒在非洲绿猴肾细胞(Vero细胞)上高效增殖的最适条件。方法将Vero细胞在50cm2细胞瓶或3000mL旋转瓶中培养成单层,以不同感染复数接种流感病毒,在不同的培养条件下孵育,取上清测病毒血凝滴度。结果当加入胰酶终浓度为40μg·mL-1时,低感染复数接种流感病毒,可获得高效价病毒液,在3000mL旋转培养瓶中流感病毒的易感性较在50cm2静置培养瓶中略高。结论建立了流感病毒在Vero细胞上高效增殖的初步方法。     

Release of Membrane-Bound Trails by Trypanosoma cruzi Amastigotes onto Modified Surfaces and Mammalian Cells

ABSTRACT. Upon incubation at 37° C onto glass coverslips coated with Concanavalin A, poly-L-lysine, or a monoclonal antibody (1D9) directed to the parasite major surface glycoprotein Ssp-4, extracellular Trypanosoma cruzi amastigotes release trails of material barely visible by light microscopy. This release is not associated with parasite movements. Immunolabeling studies confirmed that the material is derived from the parasite's membrane since thin section through samples labeled with 1D9 revealed that the trails are membrane-bound structures. Scanning electron microscopy showed that the ∼0.1-μm thick trails of material emerging from the amastigotes can be uniform or beaded, indicating a tendency to vesiculation. The trails are preferentially released from the flagellar pocket region and/or at the opposite posterior end of the parasite body, and seem to be devoid of microtubules. The release is time and temperature-dependent and fixed parasites do not form trails. All attempts to inhibit trail release using drugs (antimycin A, sodium azide, cytochalasin D, nocodazole, genistein, staurosporine, EGTA) failed. The observation of trails associated with intracellular parasites and amastigotes invading Vero cells suggests that this is probably a physiological process.     

乙型肝炎病毒重组DNA转化Vero绿猴肾传代细胞分泌乙型肝炎病毒表面抗原

使用首尾相连的乙型肝炎双拷贝DNA重组质粒pTHBV-1,以pSV2-gpt(Eco-gpt)为选择标记基因,共转化CV-1绿猴肾非营养缺陷型细胞,用霉酚酸、黄嘌呤(MX)作选择培基,选出分泌表面抗原(HBsAg)的细胞系,但HBsAg分泌量较低,见前报告。Christman等人报告,利用二氢叶酸还原酶(dhfr)基因和乙肝重组DNA质粒共转化NIH     

A novel serum-free medium for the cultivation of Vero cells on microcarriers

Summary A novel serum-free medium for the cultivation of Vero cells on microcarriers was developed,which composed of the 1:1 mixture of Dubecco's Modified Eagle Medium: Nutrient Mixture F12, bovine serum albumin(BSA) or human serum albumin(HSA), epidermal growth factor(EGF), gelatin and Dbiotin. Both BSA and EGF were effective on cell growth, adhesion and spreading. Further addition of gelatin and biotin led to the enhanced cell adhesion and spreading without growth promoting activity. The serum-free medium was suitable for the cultivation of vero cells on several different microcarriers with cell density reached over 3×l0⁶cells/ml.     

Vero 细胞流感病毒的大规模培养和纯化

目的：采用反应器技术以细胞为基质培养流感病毒,并探索其纯化工艺。 方法：采用5L反应器无血清条件下以Vero细胞为基质培养流感病毒,病毒收获后,经灭活、澄清,采用阴离子柱去除宿主DNA,亲和柱浓缩流感病毒,最后采用分子筛三步层析法进行纯化。结果：整个纯化工艺HA回收率达102%,蛋白降低率为95.3%,宿主蛋白降低率为99.77%,DNA的降低率为99.99%。结论 本研究成功建立了一种细胞流感病毒的纯化工艺。     

SARS—CoV感染Vero E6细胞诱导细胞凋亡

为了确定SARS冠状病毒(SARS—CoV)感染Vero E6是否引起细胞凋亡,我们利用细胞DNA琼脂糖电泳,感染细胞的间接荧光染色和Hoechst 33258细胞核染色,以及流式细胞仪分析等方法证明了SARS-CoV感染的Veto E6具有典型的凋亡细胞学和生物化学特征。实验证明具有细胞凋亡特征的所有细胞均为处于感染晚期的细胞。表现明显细胞病变(CPE)的细胞大多已经出现核质凝缩或形成凋亡小体进入细胞凋亡的过程。可以断定SARS-CoV感染Vero E6细胞诱发了细胞凋亡。     

用Vero细胞制备马抗狂犬病血清抗原

以Vero细胞为基质制备马抗狂犬病血清用抗原,以期建立有效、经济、简便的抗原制备方法。用含10％马血清营养液对Vero细胞作适应性培养,接种狂犬病毒,以含1％～3％马血清营养液作维持液培养病毒,于第5,8天收获病毒液,经灭活、浓缩、离心等制成抗原。第5,8天收获病毒滴度可稳定在7．010gLD50／mL以上,灭活抗原具良好的抗原性,用NIH法测定效价达6．0IU／mL以上,可用作抗原生产抗狂犬病血清。     

Standardization and validation of Vero cell assay for potency estimation of diphtheria antitoxin serum

Diphtheria toxin has the capacity to block protein synthesis in cultured mammalian cells, and thus causing cell death. This capacity of diphtheria toxin was utilized for in-vitro neutralization test to determine antibody titer, using Vero cells, which have been found to be susceptible to diphtheria toxin. In the present study, a Vero cell assay was standardized and validated for potency estimation of diphtheria antitoxin serum (DATS). The results obtained by Vero cell assay were compared with in-vivo biological assay. High degree of correlation (+0.98) was found between in-vivo biological assay and in-vitro Vero cell assay. The assay has also been found to be effective in determining the rising antibody titer in the equines inducted in DATS production. The present study indicated that although biological assays hold the key for final potency estimations till date but in the future scenario in-vitro Vero cell assay may be a good alternative to in-vivo biological assay.     

Preparation of a purified, inactivated Japanese encephalitis (JE) virus vaccine in Vero cells

An attenuated Japanese encephalitis (JE) virus SA14-14-2 (PDK) was adapted to Vero cells, a continuous cell line that has been licensed for human vaccine production, by serial passages. The resulting virus was purified by tangential flow ultrafiltration followed by sucrose density gradient ultracentrifugation, giving 2.3 mg purified virus per liter of culture supernatant. Treatment with 0.05% formalin for 4 days at 22 °C completely inactivated viral infectivity while preserving its antigenicity. The purified, inactivated JE virus was formulated with alum hydroxide and administered to mice by intraperitoneal route. In terms of its ability to induce anti-JE neutralizing antibody and to protect the immunized animal against neurovirulent virus challenge, the purified, inactivated JE virus formulated with alum was equivalent to the exiting commercial mouse brain-derived vaccine (JE-VAX, Aventis Pasteur Inc.).     

用Vero细胞制备马抗狂犬病血清抗原

以Vero细胞为基质制备马抗狂犬病血清用抗原,以期建立有效、经济、简便的抗原制备方法.用含10%马血清营养液对Vero细胞作适应性培养,接种狂犬病毒,以含1%～3%马血清营养液作维持液培养病毒,于第5,8天收获病毒液,经灭活、浓缩、离心等制成抗原.第5,8天收获病毒滴度可稳定在7.0logLD50/mL以上,灭活抗原具良好的抗原性,用NIH法测定效价达6.0IU/mL以上,可用作抗原生产抗狂犬病血清.