HAV病毒液的3种浓缩方法比较

目的：对HAV病毒液的3种常见浓缩方法进行分析比较,为HAV病毒研究及规模化疫苗生产提供参考。方法：使用MILLIPOREPELLICON超滤、PEG6000沉淀、蔗糖．甘油垫三种方法对纯化HAV病毒液进行浓缩,用ELISA方法对浓缩液进行抗原滴度检测,计算不同浓缩方法的回收率。结果：HAV病毒液经过7次超滤循环浓缩,平均回收率为86％;PEG浓缩方法回收率平均72．5％;蔗糖．甘油离心浓缩方法平均回收率53．3％。结论：蔗糖／甘油超离心法,集纯化浓缩一体,适用于样品量较少,需要高浓度样品的试验;PEG浓缩得率适中,操作简单,应用范围较广;超滤膜浓缩在大规模疫苗生产或样品量较大时适用,但需控制样品浓度及浓缩倍数不能太高。以免样品损失。     

水体病毒浓缩方法的建立和优化研究

采用氯化钙(CaC l2)、聚乙二醇(PEG,pH7.0)、聚乙二醇(PEG,pH11.5)、三氯化铝(A lC l3)沉淀、Am icon Utcra超滤离心装置和硝酸纤维素吸附膜6种浓缩方法,浓缩人工添加于水体的1型脊髓灰质炎疫苗病毒(PV1),并对浓缩实验条件进行选择和优化。结果表明,CaC l2和聚乙二醇(pH7.0)沉淀法适用于浓缩大容量水体中的病毒,而超滤离心管浓缩法适用于小容量水体,这3种浓缩方法的病毒回收率均达到100%。     

Vero细胞狂犬病疫苗在凝胶柱层析工艺中样品浓度对纯化效果的影响

为研究浓缩Vero细胞狂犬病疫苗蛋白浓度对纯化效果的影响。用超滤浓缩法对狂犬病疫苗进行不同倍数的超滤浓缩,将浓缩后不同倍数的样品分别用凝胶柱层析法进行纯化,结果样品浓度在40mg/ml以下时,狂犬病毒收集液中杂蛋白去除率可达99%以上,小牛血清含量在25-37.5ng/ml之间;浓缩超过40mg/ml时,并随着样品浓度的增加,狂犬病毒收集液中杂蛋白去除率逐渐下降,且有病毒丢失,因此采用凝胶柱层析法纯化狂犬病疫苗,蛋白浓度应低于40mg/ml。     

狂犬病疫苗蔗糖密度梯度离心纯化工艺开发

目的:开发冻干人用狂犬病疫苗(鸡胚成纤维细胞)的连续流蔗糖密度梯度离心纯化工艺。方法:分别比较两种不同初始蔗糖浓度和不同上样速度对纯化效果的影响,初步确定纯化工艺;通过多批次实验确定样品收集范围;比较不同浓缩倍数条件下杂质去除和抗原回收情况,确定合适的收获液浓缩比例;比较不同批次样品纯化后的杂质去除率和重复性,判定本纯化工艺的稳定性。结果:选取60%作为初始蔗糖浓度,在上样速度为150～200ml/min时,可以有效地对10倍浓缩的病毒收获液进行纯化;卵清蛋白、牛血清白蛋白和庆大霉素去除率分别达到99%,95%和95%,且工艺具有极好的稳定性。结论:开发的连续流蔗糖密度梯度离心技术可以作为冻干人用狂犬病疫苗(鸡胚成纤维细胞)的产业化纯化工艺。     

脊髓灰质炎减毒活疫苗的高效纯化及特性研究

应用Q-Sepharose Fast Flow对Vero细胞基质制备的I型口服脊髓灰质炎减毒活疫苗悬液进行纯化.病毒液经滤过澄清和超滤浓缩,获得85%的病毒感染性滴度回收率,而经Q-Sepharose F.F.纯化的病毒悬液,病毒感染性滴度回收率达100%.纯化后的病毒液用α-32P dATP标记DNA探针膜杂交法测定,宿主Vero细胞基质DNA残余含量远低于100 pg/剂量的标准;rct/40特征、病毒形态及病毒衣壳蛋白组份等生物学性状无显著变化.研究结果提示,Q-Sepharose F.F.是Vero细胞制备口服脊髓灰质炎减毒疫苗的理想纯化材料.     

阴离子交换色谱纯化甲型肝炎病毒(YN5株)的研究

对阴离子交换色谱纯化HAV的合适条件进行了探索。先使用“试管法”研究DEAE Sepharose Fast Flow凝胶结合HAV及病毒解离的条件,然后分别使用线性阶段洗脱和阶段梯度洗脱在柱色谱上进行了HAV的纯化。结果表明经过阴离子交换色谱纯化得到的病毒保持有抗原性和免疫原性,HAV抗原回收率大于85％,杂蛋白去除率大于80％,纯化的病毒样品中的内毒素与宿主DNA的含量也大大降低,证明阴离子交换色谱可用于HAV疫苗的纯化。     

重组人尿激酶原纯化中浓缩方法的比较

对尿激酶原纯化中的浓缩方法进行了探索,先后采用了3种不同的方法,即超滤、CM-阳离子柱、疏水色谱柱浓缩。结果表明,超滤只适合于大量样品的浓缩,样品量较小时损失较大,而且机械作用较强会使部分产品分解;CM-阳离子枉浓缩回收率较高,但得到的产品盐浓度高,还需要经过脱盐处理,增加了操作步骤,而疏水柱浓缩方法较为理想,经它浓缩后的产品用SDS-PAGE分析,非还原条件下,纯度在98％以上,还原条件下单链占90％以上,浓缩16倍,尿激酶原浓度达到3mg/ml以上。     

小量快速浓缩病毒蛋白的研究

采用甘油透析浓缩及ＰＥＧ－６０００沉淀相结合的方法,对细胞病毒培养液进行处理,获得了较为纯化的病毒蛋白。该方法适用于病毒蛋白小量快速分析。纯化的病毒蛋白回收率约为５３μｇ／ｍｌ。     

噬菌体浓缩方法的比较

目的：对不同噬菌体浓缩方法进行比较。方法：采用PEG沉淀、PEG反透析、阴阳离子结合树脂、超滤等5种方法对T4、T7、N4等3类噬菌体进行浓缩对比试验;在此基础上,用浓缩效果较好的方法对土壤和河道污水样本中的噬菌体进行浓缩,观察浓缩效果。结果：上述5种方法对3类噬菌体均有浓缩效果;超滤的浓缩效果最好,其次是PEG反透析,PEG沉淀没有很好的浓缩效果;河水样本中噬菌体的回收率比土壤样本高。结论：可通过PEG反透析、超滤或两者相结合的方法,得到高浓度的有活性的噬菌体。     

水体病毒钙离子絮凝浓缩新方法研究

研究建立了一种从水体中浓缩病毒的新方法,即钙离子絮凝-柠檬酸缓冲液洗溶法,该方法的要点是先用一定量的钙离子溶液和钙离子絮凝剂絮凝水体中的病毒,再用pH5.0的0.3mol/L的柠檬酸缓冲液洗溶,然后再进一步超滤浓缩。此法可方便地将水体中的病毒浓缩10000倍以上。应用该法分别对人工播种于饮用水中的f2噬菌体和脊髓灰质炎疫苗病毒(PV1)进行了浓缩,结果发现f2噬菌体的平均回收率达96%,而PV1的回收率为100%,均显著高于阳电膜过滤法(P<0.05)。该方法快速、简便、有效。     

Continuous-Flow Ultracentrifugation of Canine Distemper Virus and Infectious Canine Hepatitis Virus

The use of a Spinco L-4 zonal centrifuge with the B-XVI continuous-flow rotor for the purification of canine distemper and infectious canine hepatitis viruses is described. Up to 68 liters of virus was processed at one time. Infectious canine hepatitis virus was found to band at 39% sucrose and canine distemper virus banded between 32 and 48% sucrose. The virus was concentrated 10-fold, and the purity of the virus, as measured by protein concentration, was increased by 99%.     

Recovery of Protective Activity in Rabies Virus Vaccines Concentrated and Purified by Four Different Methods

Rabies vaccines concentrated by ultrafiltration, zinc acetate precipitation, ammonium sulfate precipitation, or aluminum phosphate gel adsorption were compared with respect to recovery of protective activity and purity, as measured by protective activity per mg of protein. Vaccine obtained by ammonium sulfate precipitation had a better recovery rate and a higher purity than those prepared by the other methods. Potent vaccines were also obtained by the zinc acetate precipitation and aluminum phosphate gel adsorption methods, whereas ultrafiltration was the least satisfactory method from the standpoint of vaccine purity. Chromatography of virus concentrated by ultrafiltration on a cellulose ion exchange column reduced the level of nonviral proteins. The protective activity data obtained for the vaccines examined in these experiments were found to correlate with the vaccine's complement fixation titer per mg of protein.     

Comparison of Methods for the Recovery of Virus Inoculated into Ground Beef

Various methods for the recovery of virus inoculated into ground beef were investigated in an attempt to develop a sensitive system that could be used to detect viral contaminants in market foods. A 100-g sample, inoculated with poliovirus 1, was suspended in 150 to 900 ml of Eagle minimum essential medium, pH 8.5, and mixed in either plastic bags or plastic cups on a mechanical shaker. The particulate materials were removed by means of cheese cloth, glass wool, woven fiber glass, or low-speed centrifugation. Large volumes of fluid were concentrated by ultrafiltration. Microbiological contamination was controlled by high antibiotic concentrations or by filtration. Quantitative plaque-forming-unit recovery of the virus was determined by utilizing an agar overlay technique on Vero cell cultures. The data indicated that from 20 to 50% of the seeded virus could be recovered from a 100-g sample of ground beef. The glass wool and woven fiber glass methods were the most effective, with recovery of approximately 50% of the inoculated virus.     

Concentrating rotavirus and recombinant enhanced green fluorescent protein fromE. coli using a temperature-sensitive hydrogel

Recombinant enhanced green fluorescent protein (EGFP) fromE. coli was concentrated 1.9 times by ultrafiltration using a temperature-sensitive hydrogel. Protein recovery and separation efficiency were 64% and 45%, respectively. Increased concentration of recombinant EGFP was confirmed by SDS-PAGE. Rotavirus was concentrated 3.2 times by ultrafiltration using a temperature-sensitive hydrogel, at 95% of virus recovery and 93% of separation efficiency. Hydrogel ultrafiltration appears to be an attractive alternative for the concentration of rotavirus and recombinant proteins fromE. coli.     

两种甲型肝炎病毒抗原快速检测方法的建立

目的建立两种甲型肝炎病毒抗原(HAV-Ag)检测试剂盒,并对其检测效果进行评价。方法生物素标记甲型肝炎病毒抗体(HAV-Ab)与辣根过氧化物酶标记亲和素联合应用建立甲型肝炎病毒抗原BA-ELISA检测法;同时使用辣根过氧化物酶标记HAV-Ab作放大系统建立双抗体夹心甲型肝炎病毒抗原ELISA检测试剂,对比两种检测方法的特异性、灵敏度及实际应用效果。结果用生物素标记甲型肝炎病毒抗体-辣根过氧化物酶标记亲和素作放大系统建立的甲型肝炎病毒抗原BA-ELISA检测法,较双抗体夹心ELISA检测方法灵敏度高1～2个稀释度;两种检测法均对10余种病毒无交叉,P/N值BA-ELISA检测法较高。结论甲型肝炎病毒抗原BA-ELISA检测法是一种灵敏度高,特异性好,方便快捷的检测方法,可广泛应用于甲型肝炎病毒研究及临床检测中。而甲型肝炎病毒抗原双抗体夹心ELISA检测法,检测灵敏度适中,操作简单,更适用于甲肝疫苗生产检定。     

Effect of freezing rates and excipients on the infectivity of a live viral vaccine during lyophilization

Lyophilization is the most popular method for achieving improved stability of labile biopharmaceuticals, but a significant fraction of product activity can be lost during processing due to stresses that occur in both the freezing and the drying stages. The effect of the freezing rate on the recovery of herpes simplex virus 2 (HSV-2) infectivity in the presence of varying concentrations of cryoprotectant excipients is reported here. The freezing conditions investigated were shelf cooling (223 K), quenching into slush nitrogen (SN2), and plunging into melting propane cooled in liquid nitrogen (LN2). The corresponding freezing rates were measured, and the ice crystal sizes formed within the samples were determined using scanning electron microscopy (SEM). The viral activity assay demonstrated the highest viral titer recovery for nitrogen cooling in the presence of low (0.25% w/v sucrose) excipient concentration. The loss of viral titer in the sample cooled by melting propane was consistently the highest among those results from the alternative cooling methods. However, this loss could be minimized by lyophilization at lower temperature and higher vacuum conditions. We suggest that this is due to a higher ratio of ice recrystallization for the sample cooled by melting propane during warming to the temperature at which freeze-drying was carried out, as smaller ice crystals readily enlarge during warming. Under the same freezing condition, a higher viral titer recovery was obtained with a formulation containing a higher concentration of sugar excipients. The reason was thought to be twofold. First, sugars stabilize membranes and proteins by hydrogen bonding to the polar residues of the biomolecules, working as a water substitute. Second, the concentrated sugar solution lowers the nucleation temperature of the water inside the virus membrane and prevents large ice crystal formation within both the virus and the external medium.     

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。