应用SDS—PAGE及光密度扫描测定肾综合征出血热纯化疫苗（？…

应用ＳＤＳ－ＰＡＧＥ后光密度扫描的方法,对兰州生物制品研究所生产的５批肾综合征出血热（ＨＦＲＳ）乳鼠脑Ⅰ型纯化疫苗进行了纯度测定,结果５批疫苗的纯度为７５．７％￣８９．８％,平均为８１．６８％,说明兰州所生产的乳鼠脑纯化疫苗纯度较好,杂蛋白含量很少。为精制纯化疫苗。     

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。     

精制狂犬病疫苗纯化方法的比较研究

地鼠肾细胞狂犬病疫苗原液经１００ ｋＤ 膜浓缩 ３０ 倍,分别选用（１）ＤＥＡＥ Ｓｅｐｈａｒｏｓｅ ＣＬ－６Ｂ离子交换层析法;（２）Ｓｅｐｈａｃｒｙ１ Ｓ－２００ ＨＲ 分子筛选层析法;（３）二次蔗糖等密度区带离心法对其进行纯化。用此三种方法各试制３ 批精制疫苗,结果表明,经ＤＥＡＥ Ｓｅｐｈａｒｏｓｅ ＣＬ－６Ｂ离子交换层析纯化后疫苗总蛋白含量减少９９％ 以上,抗原比活性提高１５９ 倍,抗原回收率达５０％ ,纯化疫苗以ＮＩＨ 法效力测定平均为５．４ ＩＵ／２ｍ ｌ;经Ｓｅｐｈａｃｒｙ１ Ｓ－２００ＨＲ 分子筛层析纯化后疫苗总蛋白含量减少 ９８％ 以上,抗原比活性提高４１ 倍,抗原回收率达６３％ ,纯化疫苗效力平均为６．２５ ＩＵ／２ｍ ｌ;经一次蔗糖等密度区带离心法纯化后疫苗总蛋白含量减少９８％ 以上,抗原比活性提高３２１ 倍,抗原回收率达４３％ ,纯化疫苗效力平均为６．１８ ＩＵ／２ｍ ｌ,三种纯化疫苗均符合Ｗ ＨＯ 规程要求。     

重组葡激酶的分离纯化

在构建出高表达,高活性的葡激酶工程菌株的基础上,对表达产物进行了分离纯化研究。经离子交换纯化后,纯度达８５％－９０％,回收率为５０％－６０％,经凝胶过滤后,纯度达９９％以上,回收率为６０％,经ＳＤＳ－ＰＡＧＥ测定,相对分子质量为１５．５＊１０＾３,比活为１．０＊１０＾５,Ｎ端氨基酸序列与献报道的相符。     

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。     

基因工程变链菌防龋疫苗的研制

本文采用我们自己构建的基因工程龋齿疫苗菌株,大罐发酵培养３批、菌体收量平均湿重为２．６７ｇ／Ｌ,表达率为４４．２７％。菌体经超声裂解,硫酸铵和链霉素粗提,再经吸附层检,凝胶过滤和离子交换柱层析等步骤纯化精提ＧＴＦ抗原３批。提纯的ＧＴＦ抗原,平均比活,提纯倍数和收率,分别为５８．３４ｕ／ｍｇ、６．０８倍和１９．２５％。此抗原经ＳＤＳ－ＰＡＧＥ电泳,分子量为５５ＫＤ,纯度达到９６．７％,并对湿度具有较好的稳定性能。且此抗原制备的疫苗免疫家兔具有较好的免疫原性     

应用流行性出血热病毒抗NP和G1、G2单抗检测感染细胞和疫苗内的不同抗原成分

应用出血热病毒的抗核蛋白（ＮＰ）和糖蛋白（Ｇ１、Ｇ２）单克隆抗体的ＥＬＩＳＡ夹心法对感染细胞培养物和灭活疫苗内的ＮＰ和Ｇ１、Ｇ２抗原成分进行检测,结果表明感染Ｖｅｒｏ－Ｅ６细胞内ＮＰ和Ｇ１、Ｇ２含量均高于细胞外培养液上清,前者的抗原滴度分别为≥５１２和２５６,后者仅为６４和１６。比较三种不同疫苗内的抗原成分,显示乳鼠脑纯化疫苗内的ＮＰ抗原最高,滴度达３２０－≥６４０,而二种细胞疫苗（沙鼠肾和地鼠肾细胞）则较低,一般在２０－８０,相反二种细胞疫苗的糖蛋白滴度则高于脑疫苗（８－３２对２－８）该方法可用于疫苗生产过程中检测ＮＰ和Ｇ抗原成分。     

雷丸凝集素的纯化及理化性质的研究

雷丸（Ｏｍｐｈａｌｉａ ｌａｐｉｄｅｓｃｅｎｓ Ｓｃｈｒｏｅｔ．）经过Ｔｒｉｓ－ＨＣｌ缓冲液浸提,硫酸铵分级沉淀,离了负,卵粘蛋白－Ｓｅｐｈａｒｏｓｅ ４Ｂ亲和层析以及Ｓｅｐｈａｃｒｙｌ－Ｓ１００分子筛等步骤,纯化得到纯度为９５％以上的雷丸凝集素（简称ＯＬＬ）。比活提高４５．８倍,活力回收２．５％。雷丸订素是单一肽链的蛋白质,分子量为１２ｋＤａ,等电点为７．５,可０被半乳糖抑制。具有热稳定性及酸碱     

肾综合征出血热乳鼠脑纯化疫苗(I型)糖蛋白及核蛋白在免疫保护中的作用

为了阐明肾综合征出血热纯化疫苗（Ｉ型）中糖蛋白（ＧＰ）、核蛋白（ＮＰ）在免疫保护中的作用,采用脾细胞转输法,分别将ＧＰ、ＮＰ、ＧＰ＋ ＮＰ免疫小鼠后的脾细胞与未经免疫的小鼠正常脾细胞（３．５×１０５ 个）经腹腔转输给乳鼠,２ ｈ 后以１００ ＬＤ５０的汉滩（ＨＴＮ）病毒（ＬＲ１ 株）进行脑腔攻击。结果表明,其乳鼠的保护率分别达７７．０％ ,３８．５％ 和９２．３％ ,而未经免疫的小鼠正常脾细胞则不能提供保护（０％ ）,揭示了ＧＰ＋ ＮＰ在抗ＨＴＮ 病毒感染中具有免疫协同作用。     

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。     

肾综合征出血热(I型)纯化疫苗防病效果及免疫持久性观察

卫生部兰州生物制品研究所生产的肾综合征出血热（ＨＦＲＳ）（Ｉ型）纯化疫苗在陕西、湖南,浙江的出血热流行区进行了人群免疫效果观察。基础免疫三针后,中和抗体阳转率平均５０％,荧光抗体阳转率为８４２６％,全程接种者２６４９２人,疑似发病１人,保护率平均为９６％。对陕西长安县不同年龄的３０人进行了免疫后２５年抗体水平观察,中和抗体阳转率为５７％,有良好的免疫效果,并具有一定的免疫持久性。     

国产与法国Merieux产伤寒Vi多糖菌苗人体接种反应及血清抗体应答比较

作者对国产伤寒Ｖｉ多糖菌苗和法国Ｍｅｒｉｅｕｘ产伤寒Ｖｉ多糖菌苗人体接种反应及血清抗体应答情况进行比较,接种后６～８小时法国菌苗的体温反应同国产菌苗相比有显著性差异（ｘ２＝５．３４７,０．２５＞Ｐ＞０．０１）,２４小时后反应消失,免后一月国产菌苗和法国菌苗Ｖｉ抗体四倍增长率分别为９１．８０％和８９．６％（ｘ２＝０．１６４,Ｐ＞０．０５）,ＧＭＴ分别为３６．９４和３５．４９（ｔ＝０．６５３,Ｐ＞０．０５）,二者均无显著性差异。     

DNA vaccination with the Hantaan virus M gene protects Hamsters against three of four HFRS hantaviruses and elicits a high-titer neutralizing antibody response in Rhesus monkeys

Four hantaviruses-Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava virus (DOBV) and Puumala virus-are known to cause hemorrhagic fever with renal syndrome (HFRS) in Europe and Asia. HTNV causes the most severe form of HFRS (5 to 15% case-fatality rate) and afflicts tens of thousands of people annually. Previously, we demonstrated that DNA vaccination with a plasmid expressing the SEOV M gene elicited neutralizing antibodies and protected hamsters against infection with SEOV and HTNV. Here, we report the construction and evaluation of a DNA vaccine that expresses the HTNV M gene products, G1 and G2. DNA vaccination of hamsters with the HTNV M gene conferred sterile protection against infection with HTNV, SEOV, and DOBV. DNA vaccination of rhesus monkeys with either the SEOV or HTNV M gene elicited high levels of neutralizing antibodies. These are the first immunogenicity data for hantavirus DNA vaccines in nonhuman primates. Because a neutralizing antibody response is considered a surrogate marker for protective immunity in humans, our protection data in hamsters combined with the immunogenicity data in monkeys suggest that hantavirus M gene-based DNA vaccines could protect humans against the most severe forms of HFRS.     

流行性感冒裂解疫苗临床免疫效果观察

兰州生物制品研究所研制的流行性感冒病毒裂解疫苗于2003年9月~12月在广西进行Ⅰ~Ⅲ临床试验,对疫苗的安全性和免疫原性进行考核评价。试验中随机选取852人(6月龄~67岁)接种试验疫苗,227人接种对照疫苗。6~36月龄的婴幼儿接种2针,每次0.25m l,间隔28天;成人接种0.5 m l。所有接种对象均未见红肿和硬结等局部反应;发生低热反应(37.1℃~37.5℃)率为3.5%,均于48小时内恢复正常。疫苗接种后易感人群的H I抗体总阳转率为100%,非易感人群的H I抗体几何平均效价增长7.1~16.8倍,抗体4倍增长率为73.1%~91.7%。证实该疫苗具有良好的安全性和免疫原性。     

Serologic diagnosis of mild, unclear and asymptomatic forms of hemorrhagic fever with renal syndrome

The use of the serological method (the indirect fluorescent antibody test) for the examination of 255 persons coming in contact with patients having hemorrhagic fever with the renal syndrome (HFRS) in the foci of this infection where outbreaks of group infection had been registered made it possible to detect mild, unclear and asymptomatic forms of HFRS in 17.7-20.9% of cases. The simultaneous titration of 317 serum samples with two antigens revealed that HFRS virus of serotype 1, similar to strain Hantaan from Korea, may cause, besides the classical forms of the disease, the development of mild and asymptomatic forms of this infection.     

Wild-Type Puumala Hantavirus Infection Induces Cytokines, C-Reactive Protein, Creatinine, and Nitric Oxide in Cynomolgus Macaques

Hantaviruses cause two severe human diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Approximately 200,000 cases are reported annually, and there is to date no specific treatment available. A major obstacle in studying the medical aspects of HFRS and HPS has been the lack of an adequate animal model. Here we show that infection of cynomolgus macaques by wild-type Puumala hantavirus resulted in typical signs of HFRS including lethargy, anorexia, proteinuria, and/or hematuria, in addition to cytokine (interleukin 6 [IL-6], IL-10, and tumor necrosis factor alpha), C-reactive protein, creatinine, and nitric oxide responses. Viral RNA was detected in plasma from days 3 to 7 postinoculation until days 24 to 28 postinoculation, infectious virus was recovered, and the virus-specific immune responses (immunoglobulin M [IgM], IgG, and neutralizing antibodies) mimicked those seen in humans. The results indicated that the monkey model will provide a valuable tool for studies of pathogenesis, candidate vaccines, and antivirals for hantavirus disease.     

Delivering vaccines to the people who need them most

Thanks to the Global Alliance for Vaccines and Immunization (GAVI), the Vaccine Fund and the Bill & Melinda Gates Foundation, the global health community has made enormous progress in providing already existing vaccines to developing countries. However, there still exists a gap to develop vaccines for which there is no market in the Western world, owing to low economic incentives for the private sector to justify the investments necessary for vaccine development. In many cases, industry has the technologies, but lacks the impetus to direct resources to develop these vaccine products. The present emergency with the Ebola vaccine provides us an excellent example where a vaccine was feasible several years ago, but the global health community waited for a humanitarian disaster to direct efforts and resources to develop this vaccine. In the beginning of 2015, the first large-scale trials of two experimental vaccines against Ebola virus disease have begun in West Africa. During the past few years, several institutions have dedicated efforts to the development of vaccines against diseases present only in low-income countries. These include the International Vaccine Institute, the Novartis Vaccines Institute for Global Health, the Hilleman Institute, the Sabin Vaccine Institute and the Infectious Disease Research Institute. Nevertheless, solving this problem requires a more significant global effort than that currently invested. These efforts include a clear policy, global coordination of funds dedicated to the development of neglected disease and an agreement on regulatory strategies and incentives for the private sector.     

两种方法检测接种流行性出血热沙鼠肾细胞灭活疫苗后人体的中和抗体

本文应用空斑减少中和试验(PRNT)和细胞病变中和试验(cPENT)两种方法对出血热沙鼠肾细胞灭活疫苗扩大人体免疫后的血清进行中和抗体水平检测。根据两种方法对总计74人份的免疫后血清检测比较结果,两种方法检测的抗体阳转率和抗体水平(GMT)。CPENT法均高于PRNT法,经统计学处理均有显著性差异。不同免疫组的中和抗体水平比较结果,注射三针的阳转率(n=10,100％)高于两针组(n=10,20—30％);接种加氢氧化铝佐剂疫苗(n=13)较接种不加佐剂的两种疫苗(n=26)的抗体水平高,阳转率为92％—100％GMT为22—69;皮下途径(n=15)和肌肉途径(n=13)注射无明显差别,阳转率分别为87—93％和92—100％,GMT分别为29—46和22—61。以上结果进一步肯定沙鼠肾细胞疫苗的人体免疫性     

重组单克隆抗体药物的工程细胞培养工艺研究

单克隆抗体(monoclonal antibody, mAb)是一类重要的基因工程生物技术衍生药物,其药物适应证包括自身免疫性疾病、移植后并发症、心血管疾病、感染性疾病和各种类型的癌症。此类适应证药物通常使用时间长、使用剂量大,因此需具备大批量生产的能力。由于mAb药物的生产成本以及产品质量控制的复杂性,要求有效且经济地提供批间一致性较好的高质量药物。中国仓鼠卵巢(Chinese hamster ovary, CHO)细胞是生物技术产业中用于生产治疗性糖蛋白最常用的真核表达宿主,也是生产治疗性抗体药物最常使用的细胞。在细胞培养过程中,工程细胞株、培养基和工艺条件是mAb药物生产中影响其质量的三个重要因素。现分别从培养基、培养参数和培养模式等对生产mAb药物的细胞培养技术作一概述。