Vero毒素—1的纯化及特性分析

从含有VT1全基因的基因工程菌中纯化出VT1。纯化的步骤包括（NH4）2SO4盐析,两次DEAE Sepharose Fast Flow柱层析。最终从4L培养物中纯化出1.5mg纯毒素,收率为8．6％,梯度Native-PAGE测定毒素的分子量为70kD,SDS－PAGE电泳表明毒素有两个亚基,分子量分别是32kD和7.7kD。对VT1的多种生物学特性进行了研究;经测定VT1对Vero细胞的半数致死量CD50为1pg,对小鼠的半数致死量LD50为18ng,引起兔肠襻积液的最小毒素量是1．25μg/肠襻。     

Vero细胞微载体不同培养方法的评价

对三种不同培养方法进行细胞生长速度、密度、营养及代谢产物浓度的比较分析,以优化和筛选最佳培养条件与方式。用同体积生物反应罐,基本培养条件相同,采用批培养、再循环培养、灌流培养三种方式进行了Vero细胞微载体（CytodexI）的周期培养。三种培养方法均达到预期效果,最终细胞密度分别为每毫升2.09×10     

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.).     

鸡传染性支气管炎病毒的RNA干扰

为探讨短的双链RNA(siRNA)对鸡传染性支气管炎病毒(IBV)增殖的干扰作用,利用软件设计siRNA 1280个,75%位于Pol基因内.通过同源比较和保守性分析,筛选到针对Pol、M、N基因的12个siRNA(每个基因3～4个)作为后选目的片段,分别在Vero细胞、9日龄SPF鸡胚上进行基因干扰试验.结果,来自Pol、N靶序列的2个siRNA在Vero细胞上及鸡胚上均对IBV增殖产生明显的干扰作用,并与siRNA剂量有一定相关性,依赖于与mRNA互补的负链siRNA存在.本研究首次证实IBV增殖过程中存在siRNA干扰现象,为利用RNA干扰(RNAi)技术控制IBV提供了新手段.     

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.     

用Vero细胞大量制备传染性法氏囊病病毒

目前传染性法氏囊病病毒疫苗主要是用原代鸡胚成纤维细胞 (ＰＣＥＦ)增殖ＩＢＤＶ进行生产。由于ＳＰＦ种蛋价格高 ,且ＳＰＦ种蛋在取得及培养过程中易被外源病原污染 ,造成产品质量的不稳定 ,生产成本很高[1] 。Ｖｅｒｏ细胞系是一种贴壁依赖性的传代细胞系 ,ＷＨＯ已经批准用Ｖｅｒｏ细胞作为载体进行病毒疫苗的生产。目前已成功地应用Ｖｅｒｏ细胞生产出脊髓灰质炎病毒疫苗和狂犬病毒疫苗[2 ] 。用Ｖｅｒｏ细胞生产传染性法氏囊病病毒疫苗也会有较好的前景。我们已完成了在Ｖｅｒｏ细胞上静止状态下增殖ＩＢＤＶ弱毒株的培养条件研究 ,而…     

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人体的血清学效果观察

为了解国产浓缩地鼠肾细胞狂犬病疫苗与法国纯化Vero细胞狂犬病疫苗接种人体后中和抗体产生情况。分别用两种疫苗接种 1 0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 (第一针接种后 6 0天 )可 1 0 0 %达到保护水平 ,法国疫苗在第一针接种后 30天 1 0 0 %达到保护水平。第一针接种后 1 4、30、6 0天时 ,前者抗体平均滴度分别是 0 0 6IU /ml、1 .0 2IU/ml、2 .0 7IU/ml;后者抗体平均滴度分别是 0 2 5IU /ml、3.2 4IU/ml、1 1 .86IU/ml,后者比前者产生抗体的滴度高 ,具有显著性差异 (P <0 0 2 )。     

Production of high titre disabled infectious single cycle (DISC) HSV from a microcarrier culture

Disabled Infectious Single Cycle (DISC) HSV-2 has been cultured in the complimentary cell line CR2 to provide high titre bulk material suitable for the purification of the virus as a live viral vaccine. CR2 cells are cultured on the microcarrier Cytodex-1 at 5 g l-1 in small scale (1 l) and larger scale (15 l) reactors. The cells are infected at an MOI of 0.01 pfu cell-1 and the culture harvested 60–72 h later. The infected cells are removed from the microcarriers by the addition of a hypotonic saline and the virus released by low-pressure disruption techniques. Virus titres achieved are compared to the standard roller bottle process. The resulting material is the starting point for the purification of the DISC-HSV virus. This revised version was published online in July 2006 with corrections to the Cover Date.     

Discovery of novel bis-oxazolidinone compounds as potential potent and selective antitubercular agents

A variety of new mono-oxazolidinone molecules by modifying the C-ring of Linezolid, a marketed antibiotic for MRSA, were synthesized and tested for their in vitro antibacterial activities against several Staphylococcus aureus, Mycobacterium smegmatis and two Gram-negative bacteria strains (Escherichia coli and Pseudomonas aeruginosa). Among them, compounds 4–7 displayed moderate antimicrobial activities. After development of a second oxazolidinone ring in the western part of the mono-oxazolidinone compounds 4–7 by a ring closure reaction with N,N′-carbonyldiimidazole (CDI), we found thus obtained bis-oxazolidinone compounds 22–25 possess excellently inhibitory activities against H₃₇Rv but poor or no effects on other test bacteria. Among them, bis-oxazolidinone compound 22 and 24 are the most potent two compounds with a same MIC value of 0.125 μg/mL against H₃₇Rv virulent strain. Compound 22 also exhibited extremely low cytotoxicity on monkey kidney Vero cells with a selective index (IC₅₀/MIC) over 40,000, which suggested bis-oxazolidinone compound 22 is a promising lead compound for subsequent investigation in search of new antitubercular agents.     

Synthesis and evaluation of new 4-oxoquinazolin-3(4H)-yl)benzoic acid and benzamide derivatives as potent antibacterial agents effective against multidrug resistant Staphylococcus aureus

Treatment of nosocomial and community acquired Staphylococcus aureus infections has become more challenging due to the egression of multi-drug resistance. This has spurred the need for rapid development of new therapeutic agents which can effectively negate the resistance mechanisms. In our current work, several new 4-oxoquinazolin-3(4H)-yl)benzoic acid and benzamide derivatives were synthesized and examined for their antimicrobial activity against ESKAP pathogen panel and pathogenic mycobacteria. In the primary screening, compounds 4a, 4b, 6′a, 6′b, 6′h, 6′i and 6′j were found to demonstrate selective and potent inhibitory activity against Staphylococcus aureus (MICs = 0.25–0.5 µg/mL). When tested against Vero cells, all the compounds were found to be non toxic possessing favourable selectivity index (SI > 10), which encouraged us for carrying out further studies. Compound 6′a (SI > 40) was tested against a number of multiple clinical strains of multi-drug resistant S. aureus and was found to exhibit potent activity, irrespective of the resistant status of the strain. Besides, compound 6′a also exhibited concentration dependent bactericidal activity and synergized with the FDA approved drugs tested. The interesting results obtained suggest the potential utility of the newly synthesized compounds for treatment of multidrug resistant S. aureus infections.