两步串联层析法纯化鼠抗人CD80单克隆抗体4E5

采用阴离子交换与凝胶过滤两步串联层析法,纯化了小鼠腹水来源的CD80阻断型单克隆抗体4E5。腹水样品经离心、过滤预处理后,在Tris-HCl缓冲溶液(pH8.0, 50mmol/L)条件下上阴离子交换柱对目的单抗进行捕集,采用0-0.5 mol/L NaCl浓度分步洗脱;含目的单抗的洗脱馏分再上凝胶过滤柱纯化,用PB缓冲溶液（pH7.2, 20mmol/L）洗脱,获得目的单抗4E5,其生物学活性高、纯度大于95%,抗体总回收率达61%。     

甲型肝炎病毒抗原蛋白的制备与分析

目的提取并制备甲型肝炎病毒(hepatitis A virus, HAV)抗原蛋白,了解、分析其主要成分和性质。方法利用人胚肺二倍体细胞培养并收获HAV,释放并初步提取病毒抗原;采用阴离子交换层析和分子筛层析等方法纯化制备HAV抗原蛋白;再利用凝胶电泳(gel electrophoresis)和Western印迹法(Western Blot)对蛋白成分和性质进行分析。结果初步提取的HAV抗原回收率为87.50%,蛋白质去除率为87.88%。纯化后的HAV抗原蛋白的电泳显示HAV抗原蛋白主要由3条蛋白带组成,相对分子质量分别为33 000、29 000和25 000;Western Blot结果显示,相对分子质量为33 000和29 000的2条蛋白带可与抗体反应。结论实验提取并制备了HAV抗原蛋白,其电泳条带的相对分子质量与HAV衣壳蛋白VP1、VP3和VP2的相对分子质量相当,其中两条蛋白带可与抗体发生免疫反应,含有主要的抗原表位。     

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。     

茶叶中酸性杂多糖的部分化学性质及降血糖活性的研究

粗老绿茶用醇提、复合纤维素酶提取、醇沉、再经弱碱性的大孔阴离子交换树脂D315柱层析,经NaCl洗脱,超滤除盐得酸性多糖ATPS。传统多糖的分离纯化一般采用价格昂贵的DEAE纤维素柱层析,很难进行工业化生产,为考察价格便宜的大孔阴离子交换树脂D315的分离纯化性能,对其分离得到的酸性茶多糖ATPS的部分化学性质进行了研究。将ATPS上DEAE52纤维素柱进一步纯化,收集DEAE52 NaCl梯度洗脱的主峰,透析,冻干,得ATPS-Ⅰ。高效凝胶渗透色谱分析发现ATPS与ATPS-Ⅰ分子量分布基本一致,气相色谱和离子色谱分析表明两者的单糖组成均以鼠李糖、阿拉伯糖、半乳糖和半乳糖醛酸为主,而且摩尔比近似。紫外光谱扫描表明ATPS不含蛋白质,红外光谱分析表明其糖苷键键型有α-型和β-型两种,单糖主要以吡喃糖苷形式存在。实验表明大孔阴离子交换树脂D315可以起到分离纯化茶多糖的作用。动物实验表明：ATPS能抑制四氧嘧啶糖尿病小鼠血糖的升高。     

发酵液中L-色氨酸分离纯化工艺研究

通过静态吸附实验,考察了温度、pH值对001×7阳离子交换树脂平衡吸附量的影响,并测定了吸附动力学曲线。通过动态实验,测定了动态吸附曲线和洗脱曲线。最后确定了001×7阳离子交换树脂分离纯化L-色氨酸的最佳工艺条件:用001×7阳离子交换树脂吸附L-色氨酸,以浓度为2 mol.L-1氨水进行洗脱,收集的流份经D315阴离子交换树脂脱色,浓缩结晶后得L-色氨酸成品,总提取率为73.0%。     

一种快速纯化蛋白的电洗脱方法

以TB24kDa蛋白为例介绍了一种快速纯化蛋白的电洗脱方法。根据作者实验室先前建立的提取苦荞种子蛋白的方法制备TB324kDa蛋白粗提物,利用阴离子交换层析和改进的电泳洗脱方法对其进行纯化。结果显示：经改进的电洗脱纯化,1mg粗蛋白町以回收得到150μg左右的目的蛋白,同收率为15．32％,纯化效果较理想。     

β—鹅膏毒肽的反相高效液相色谱分离纯化

用反相高效液相色谱,以0．02mol/L醋酸铵—乙腈为流动相的梯度洗脱模式,在295nm吸收值的条件下,灰花纹鹅膏菌Amanita fuliginea的肽类毒素可以被成功的分离和纯化。单个肽类毒素的鉴定是用反相高效液相色谱和质谱同时进行。用这一方法可从灰花纹鹅膏菌中分离纯化出β-鹅膏毒肽(β-amanitin),产量可达到：1158μg／g(干重),产品纯度达98％以上,回收率为95．3％。β-鹅膏毒肽的分子量为919．3Da。这个方法可用于其它鹅膏菌肽类毒素的分离纯化。     

丙型肝炎病毒核心重组抗原C27的纯化

高效表达了ＨＣＶ核心区基因抗原之后,对表达蛋白Ｃ２７进行了纯化,经研究,重组蛋白是以包涵体形式存在于宿主菌内的。Ｃ２７重组蛋白分别经过包涵体洗涤、ＤＥＡＥ阴离子交换层析和Ｓ－２００分子筛两步柱层析纯化之后,纯度大于９５％,纯化得率为５３．２％,总回收率为１７．９％,纯化工艺流程简单、得率高,适合向规模化生产发展。     

辛酸沉淀结合阴离子交换层析纯化马IgG的研究

目的以辛酸沉淀结合离子交换层析纯化破伤风抗毒素马免疫血浆,获得高质量的马IgG,为抗毒素F(ab')2的制备奠定基础。方法通过对辛酸沉淀马血浆过程中的pH、辛酸浓度以及血浆稀释倍数的实验设计(DoE),研究不同条件对IgG纯度、效价、比活性、浊度以及过滤速度的影响,确定各工艺参数的可操作区间,并结合Capto DEAE阴离子交换层析以流穿模式进一步纯化IgG。结果马血浆经一步辛酸沉淀获得的IgG纯度(SDSPAGE)大于92%、分子排阻色谱(SEC)纯度大于95%,比活性较血浆提高2.14±0.29倍;辛酸沉淀后的IgG样品经阴离子交换层析,可有效去除聚合体以及小分子杂质,将纯度提高至95%(SDS-PAGE)和98%(SEC)。结论马血浆经辛酸沉淀和阴离子交换层析可获得高纯度、高比活的IgG。     

重组人促红细胞生成素纯化工艺的优化

采用堆积床生物反应器,用无血清培养基培养分泌重组人促红细胞生成素（rhEPO）的工程细胞株ZK9703.所收集的上清,采用阴离子交换层析－反相层析－分子筛层析三步纯化工艺路线,分别用Q－Sepharose XL-C4-S-200（方法Ⅰ）和DEAE Sepharose FF-Source-S-200(方法Ⅱ）纯化3批产品,所得EPO纯度达98％以上,体外比活性大于1.3^10^5IU/mg。方法Ⅰ、方法Ⅱ纯化过程的EPO体外活性回收率分别为23.56%和28.57%。本纯化方法Ⅱ工艺纯化日程短,分离效果好,EPO体内、体外活性回收率较高,更适合于大规模生产重组人促红细胞生成素。     

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。     

Evidence of Hepatitis A Virus Person-to-Person Transmission in Household Outbreaks

The person-to-person transmission of the hepatitis A virus primarily occurs in enclosed spaces, particularly in the presence of inadequate hygiene conditions and a high proportion of susceptible individuals. Thus, intimate family contact stands out as a risk factor for HAV infection dissemination. The present study aimed to evaluate the occurrence of household HAV transmission. Blood samples were collected from patients with hepatitis A (index cases) and their family members (contacts) that were referred to an ambulatory care clinic specializing in viral hepatitis. A total of 97 samples were collected from 30 families with a confirmed hepatitis A case (index case). Serological and molecular techniques for the diagnosis of hepatitis A were conducted on all samples. HAV infection (anti-HAV IgM + and/or HAV RNA +) was detected in 34.3% (23/67) of the contacts; 34.3% (23/67) of the contacts were immune to HAV, and 31.4% (21/67) were susceptible. In the household contacts, HAV immunity was significantly associated with older age; susceptibility to infection and HAV infection were associated with younger age. Household outbreaks were detected in 16/30 families studied. Co-circulation of subgenotypes IA and IB was found in the household outbreaks, and person-to-person transmission was evidenced in six of the household outbreaks, with 100% homology between the index case and contact strains. The results demonstrated the relevance of HAV household transmission, reaffirming the need for hepatitis A vaccine administration in susceptible contacts and effective infection control procedures to prevent the extension of household outbreaks.     

中国五省市甲型肝炎病毒基因分型的研究

为了解甲型肝炎（甲肝）病毒（ＨＡＶ）在中国几个城市的基因型分布,选择浙江杭州、江苏启东、安徽铜陵、云南昆明和上海市等的甲肝病人粪便标本或血清标本,以逆转录－套式聚合酶链反应（ＲＴ－ｎＰＣＲ）扩增合成ＨＡＶ ＶＰ１／２Ａ交接区基因区,并进行直接核苷酸序列分析和差异比较。结果表明,从这些城市甲肝病人分离到的１７株ＨＡＶ株均属基因Ⅰ型,为ＩＡ和ＩＢ亚型;所有ＨＡＶ株间核苷酸差异均小于１５％,但约５０％Ｈ     

TaqMan实时荧光定量RT-PCR检测甲型肝炎减毒活疫苗病毒含量的研究

目的：建立一种快速定量检测甲型肝炎减毒活疫苗病毒含量的实时荧光定量RT-PCR方法。方法对Gen-Bank中登陆的甲型肝炎减毒活疫苗株（ L-A-1）和其他甲型肝炎病毒基因组全序列比较分析,根据其高度保守的5′端非编码区设计针对甲型肝炎减毒活疫苗株特异性引物与探针,对荧光定量RT-PCR反应条件进行优化,检测该方法的特异性和灵敏性,并对甲型肝炎减毒活疫苗病毒含量进行定量检测。结果该方法对甲型肝炎减毒活疫苗株高度特异,扩增片段为207 bp,不与其他肠道病毒发生非特异性反应。在104 CCID50/管～10-1 CCID50/管之间有良好的扩增曲线,检测的灵敏度可达0．1CCID50～0．01CCID50,比普通RT-PCR高100倍。结论该方法具有快速、灵敏、特异、重复性好等优点,可应用于甲型肝炎减毒活疫苗生产过程中病毒含量滴度测定及指导疫苗成品的配制。     

Effect of the content of hepatitis A antibodies in immunoglobulin preparations on the effectiveness of the immunoglobulin prophylaxis of hepatitis A

The results of a strictly controlled experiment showed that prevention of hepatitis A by the injection of immunoglobulin with hepatitis A virus (HAV) antibody titer 1:10000 was 3 times as effective as that achieved with immunoglobulin containing HAV antibodies in titer 1:2500. It is recommended to determine the level of specific HAV antibodies in immunoglobulins and to use immunoglobulins with a high level of HAV antibodies for prevention of hepatitis A.     

国产甲型肝炎灭活疫苗用于儿童免疫效果研究

为了探讨国产甲型肝炎灭活疫苗在儿童中应用的免疫效果,选择2～15岁抗-HAV阴性健康易感儿童91名作为接种对象,采用0、6程序接种国产甲型肝炎灭活疫苗250U／剂,观察免疫后的局部反应和全身反应,并于全程免疫后一个月检测抗-HAV阳转率和抗体GMT。结果91例观察对象在初免和加强免疫后均未见即时副反应,只在8～72小时内出现轻微的一过性局部和全身反应。全程免疫后一个月抗-HAV阳转率为100％。抗体GMT为14 407mIU／ml。国产甲肝灭活疫苗在儿童中应用具有良好的安全性和免疫原性,采用0、6个月程序可获得高滴度抗体。     

Shifting seroepidemiology of hepatitis A in Japan, 1973-2003

BACKGROUND: Hepatitis A infection is caused by hepatitis A virus (HAV) contracted through fecal-oral transmission. Life-long immunity is conferred after infection. Improved sanitary conditions have generally resulted in a significant decline in the incidence of hepatitis A. However, a low incidence of infection results in increased HAV susceptibility. The present study investigates the prevalence of anti-HAV antibody and clarifies the current HAV status and HAV susceptibility in Japan at 2003. METHODS: A total of 2,430 serum specimens collected during 2003 from Japanese individuals ranging in age from 0-92 years, were tested for anti-HAV antibody using an inhibition enzyme linked immunosorbent assay. All specimens were obtained from the WHO and the National Serum Reference Bank/National Institute of Infectious Diseases, Tokyo, Japan. RESULTS: The overall seroprevalence was 12.2%. Anti-HAV antibodies were rarely detected in individuals between 0-44 years of age. Starting from the age of 45-49 years, seropositivity gradually increased through age 65 years and above. Seroprevalence was not affected by gender, and geographic distribution did not affect age-specific seroprevalence until the age of 60 years. CONCLUSIONS: HAV susceptibility in Japan is increasing annually. Particularly, the prevalence of anti-HAV antibody in individuals older than 50 years in 2003 was 50.3%, which is significantly lower than that of corresponding studies in 1994 (74.3%), 1984 (96.9%) and 1973 (96.9%). The growing susceptible population of advanced age results in more frequent HAV infection among them. The surveillance of anti-HAV antibody prevalence is useful for implementing preventive measures and for controlling the spread of HAV.