应用快速HEPES蚀斑法检测狂犬病毒

应用快速ＨＥＰＥＳ蚀斑法检测狂犬病毒邵益斌,顾勤,曾蓉芳（卫生部上海生物制品研究所;上海２０００５２）关键词ＨＥＰＦＳ蚀斑法,ＭＣ蚀斑法,小鼠脑内毒力滴定,狂犬病毒蚀斑法作为滴定活性病毒效价常用的比较精确的方法早被广泛应用。国内外学者已经建立并应用狂...     

Beagle狗肾细胞及用于制备乙脑活疫苗的研究

Ｂｅａｇｌｅ乳狗（５－１０日龄）肾块用热消化法制成细胞批放液氮保存,检定合格后做下列试验：（１）复苏培养长满单层后更换维持液,其细胞能维持４天以上,克服了常规消化培养细胞维持时间短（３６小时）的缺陷。（２）传代１４－２株病毒时,１代病毒的滴度即达６．０４－６．２４ＬｏｇＰＦＵ／ｍｌ,１代后的各代（至１１代）病毒滴度无明显提高。（３）该细胞接种１４－２株病毒时不产生明显的破坏性病变（ＣＰＥ）,在收毒前采取冻溶１次比冻溶前的病毒滴度提高０．２７－０．５ＬｏｇＰＦＵ／ｍｌ。以ＢＤＫ细胞传１代的１４－２株病毒作生产毒种,收毒前冻溶１次等工艺法制备五批疫苗,全面检定结果均符合《乙脑活疫苗规程》的质量标准。其中病毒滴度为６．０９－６．２４ＬｏｇＰＦＵ／ｍｌ;免疫效价（ＩＤ５０＝ｍｌ）为１．９－４．０×１０－５,与相同滴度的原毒株１４－２ＰＨＫ苗对照无明显差异（ｔ＝０．９６８Ｐ＞０．０５）,表明其免疫原性与原毒株相似。     

人主动脉硫酸乙酰肝素蛋白聚糖对培养人的脐静脉内皮细胞生长的影响

为探讨硫酸乙酰肝素蛋白聚糖（ＨＳＰＧ）对内皮细胞生长的作用,用解聚提取及离子交换柱层析法分离出人主动脉ＨＳＰＧ,用倒置显微镜、细胞计数、及￣３Ｎ－ＴｄＲ参入观察其对培养的第一代人脐静脉内皮细胞（ｈＵＶＦＣ）生长的影响。结果发现：（１）倒置显微镜下观察,加入ＨＳＰＧ（１．７０μｇ已糖醛酸／ｍｌ）的ｈＵＶＥＣ生长密度高于对照组（未加ＨＳＰＧ）．（２）随着培养时间增加（２４,４８及７２ｈ）．根据细胞计数计算出同一剂量的ＨＳＰＧ（１７．０μｇ已糖醛酸／ｍｌ）对ｈＵＶＥＣ的促增殖％增高（分别为１４％,３０％及３７％）。（３）随着加入ＨＳＰＧ浓度的升高（４．３,８．５及１７．０μｇ已糖醛酸／ｍｌ．培养７２ｈ）．根据￣３Ｈ－ＴｄＲ参入计算出ＨＳＰＧ对ｈＵＶＥＣ的促增殖％亦增高（分别为４９％,７１％及９８％）。故人主动脉ＨＳＰＧ对培养的人脐静脉内皮细胞有促增殖作用。     

用ELISA法检测2.5S鼠神经生长因子

鼠颌下腺提纯的２５ＳＮＧＦ免疫家兔,获得兔抗ＮＧＦ抗体,研制出鼠２５ＳＮＧＦＥＬＩＳＡ检测试剂盒,该试剂盒灵敏度小于１ｎｇ／ｍｌ,在６７０～０８４ｎｇ／ｍｌ范围内,线性良好,ｒ＝０９９。与大鼠、小鼠及人血浆无非特异反应,在大鼠血浆中,ＮＧＦ样品回收率在９１％～１０７％之间,变异系数小于１０％（ｎ＝４）。结果表明：本试剂盒操作简便,灵敏度高,特异性强,适合药代动力学研究及生产过程中的ＮＧＦ检测。     

脐带血清在骨髓造血祖细胞培养中的效应

目的：探讨人脐带清（ＣＢＳ）在骨髓造血祖细胞培养中的效应。方法：用人骨髓细胞进行ＣＦＵ、ＧＭ、ＶＦＵ－Ｅ、ＢＦＵ－Ｅ、ＣＦＵ－ＧＥＭＭ培养。结果：ＣＢＳ能直接刺激骨髓细胞ＣＥＵ－ＧＭ的形成。与血型相同害无关。四人份以上的混合ＣＢＳ（ＭＣＢＳ）,刺激活性高且稳定。１０％ＭＣＢＳ相当于６５．６μｇ／Ｌ ＧＭ－ＣＳＦ、０．２３、０．３、０。．４６ｋＵ／Ｌ ＥｐＯ对ＣＦＵ－ＧＭ、ＣＦＵ－Ｅ、ＢＦＵ－Ｅ、Ｃ     

离子对反相HPLC测定小鼠骨髓BFU—E,CFU—E中PRPP合成酶活性

采用离子对反相高效液相色谱法（ＩＰｒＨＰＬＣ）的单液等度洗脱,测定了小鼠骨髓红系爆增性集落形成单位（ＢＦＵ－Ｅｓ）与红系集落形成单位（ＣＦＵ－Ｅｓ）的ＰＲＰＰ合成酶活性。方法学研究表明,加入离子对试剂硫酸氢四丁基铵（ＴＢＡＨＳ）后,ＡＤＰ谱峰分离完全,其反应增加量易于计算。本法的批内变异系数平均值为－２．３０％￣－１．０６％与１．０６％￣２．３０％,平均相对偏差为０．８１％￣１．７４％,回收率为９     

表皮生长因子对离体大鼠肺动脉及肺动脉平滑肌细胞作用的研究

本研究着重探讨表皮生长因子（ＥＧＦ）对大鼠肺动脉的收缩作用及对肺动脉平滑肌细胞分裂增殖的影响。浓度为１×１０－９－１×１０－７ｍｏｌ／Ｌ的ＥＧＦ可引起大鼠肺动脉剂量依赖性收缩（ｒ＝０．９６８,Ｐ＜０,００１）,其Ｅｍａｘ为１００．６ｍｇ,ＥＣ５０为１１．９６ｎｍｏｌ／Ｌ。在同时存在０．５％胎牛血清（ＦＣＳ）时,ＥＧＦ能促进平滑肌细胞的３Ｈ－ＴｄＲ参入率,该作用与剂量呈正相关（ｒ＝０．８２３,Ｐ＜０．０５）,其ＥＣ５０为６．５×１Ｏ－１２ｍｏｌ／Ｌ。１×１０－９ｍｏｌ／Ｌ的ＥＧＦ＋０．５％ＦＣＳ能产生与１０％ＦＣＳ相当的促细胞分裂增殖能力（在培养的第１,３,５,７天,二者促分裂增殖能力相差不明显,Ｐ均＞０．０５,第９天时,前者大于后者,Ｐ＜０．０５）。１×１０－９ｍｏｌ／ＬＥＧＦ单独存在时对平滑肌细胞未显示出明显的致分裂活性。上述作用提示ＥＣＦ在某些肺血管病变如缺氧性肺动脉高压中可能有一定意义。     

眼镜蛇毒细胞毒素的抗肿瘤作用

目的　研究眼镜蛇毒细胞毒素（ Ｃ Ｖ Ｃ Ｔ Ｘ）的体内抗肿瘤作用。方法　小鼠皮下、腹腔接种 Ｓ１８０ 、 Ｅ Ａ Ｃ后, 于接种部位注射不同剂量的 Ｃ Ｖ Ｃ Ｔ Ｘ, 每天 １ 次, 连续１０ 天, 观察瘤重抑制率和生命延长率。结果　适当剂量（０２～０８ｍ ｇ／ｋｇ） 的 Ｃ Ｖ Ｃ Ｔ Ｘ 能明显抑制肿瘤的生长 （ Ｐ＜ ００１）,小鼠的存活时间明显延长（ Ｐ ＜ ００１）。结论　 Ｃ Ｖ Ｃ Ｔ Ｘ 在小鼠体内对 Ｓ１８０ 、 Ｅ Ａ Ｃ有明显地抗肿瘤作用。     

用蚀斑法进行麻疹疫苗的病毒滴定

按世界银行中国疫苗项目对麻疹疫苗检定的要求,建立了麻疹疫苗病毒滴定的蚀斑方法。以蚀斑法对Ｈｕ１９１株生产的冻干麻疹活疫苗样品进行了测定。结果发现,Ｈｕ１９１株生产的麻疹疫苗在琼脂和甲基纤维素覆盖下所形成的蚀斑大小及形状不同,但滴度无显著性差异;在室温和３７℃吸附的滴度无显著性差异,但细胞在３７℃生长较好;在本实验条件下,蚀斑滴定和ＣＣＩＤ５０测定的滴度呈正相关,蚀斑滴定相对较敏感;采用国产６孔聚苯乙稀培养板,Ｖｅｒｏ细胞单层,在３７℃吸附,用甲基纤维素覆盖,经结晶紫及甲醛固定染色后计数空斑,操作简便易行,结果特异敏感,重复性好,可用于麻疹疫苗病毒滴定     

DETECTION OF INFECTIOUS VIRUS AND VIRAL RNA IN THE MYOCARDIUM OF MICE INFECTED EXPERIMENTALLY WITH COXSACKIE VIRUS’B

ＤＥＴＥＣＴＩＯＮＯＦＩＮＦＥＣＴＩＯＵＳＶＩＲＵＳＡＮＤＶＩＲＡＬＲＮＡＩＮＴＨＥＭＹＯＣＡＲＤＩＵＭＯＦＭＩＣＥＩＮＦＥＣＴＥＤＥＸＰＥＲＩＭＥＮＴＡＬＬＹＷＩＴＨＣＯＸＳＡＣＫＩＥＶＩＲＵＳ’Ｂ３ＸｉａｏｍｅｉＯｕｙａｎｇ,ＨｏｎｇｙｉＺｈａｎ...     

ELISA法检测乙型脑炎减毒活疫苗中卡那霉素和庆大霉素的残留量

用ELISA试剂盒检测乙脑减毒活疫苗中卡那霉素和庆大霉素的残留量。以间接竞争ELISA法检测线性范围内加入高、中、低3种浓度的卡那霉素和庆大霉素,测定其在疫苗稳定剂、疫苗稳定剂10倍稀释溶液、试剂盒稀释液中的回收率,以及在10倍稀释乙脑减毒活疫苗中的回收率。高、中、低浓度的卡那霉素在疫苗稳定剂、疫苗稳定剂10倍稀释溶液、试剂盒稀释液中的回收率为101.40%～124.80%之间;原倍疫苗稳定剂对庆大霉素的测定有明显干扰,在原倍疫苗稳定剂中低浓度和中浓度的庆大霉素的回收率高达2280%和575%,但其它测定条件下回收率在90%～125%之间。在10倍稀释的乙脑疫苗中加入一定量的卡那霉素和庆大霉素,回收率分别为124%和103.25%。用10倍稀释法测定1人份规格的乙脑减毒活疫苗17批、5人份规格的乙脑减毒活疫苗19批。乙脑减毒活疫苗10倍稀释后,可用ELISA试剂盒检测卡那霉素和庆大霉素残留量。     

微量板细胞病变法滴定水痘疫苗的研究

建立了用微量板细胞病变法滴定水痘疫苗的实验方法。将适宜稀释度的病毒与2BS细胞混种于96孔板培养,种毒后第7天观察孔内细胞病变情况,以karber法计算疫苗滴度,并与蚀斑法进行比较。实验表明,微量板细胞病变法滴定水痘疫苗,结果稳定性好,精密度高,较现行的蚀斑法操作简便、误差小,能够较好的反映出水痘的真实滴度,可作为一种较为简便的滴定水痘疫苗的方法。     

乙型脑炎减毒活疫苗(SA_(14)-14-2)在豚鼠体内保护作用的免疫机制的研究

本实验将乙脑减毒活疫苗ＳＡ_（１４）－１４－２株以不同疫苗病毒量（３．８７ＰＦＵ／ｍｌ和５．８７ＰＦＵ／ｍｌ）分别一次免疫豚鼠,观察其对强毒攻击后抑制毒血症和抗体形成的能力。结果显示疫苗（５．８７ＰＦＵ／ｍｌ）免疫组豚鼠攻击前虽然中和抗体阴性或很低,但经攻击感染后不同时间内均未出现病毒血症,对照组豚鼠则于第２,３,４天全部出现病毒血症。表明一次活疫苗免疫后能有效地抑制病毒血症的产生。免疫后３０天虽然免疫组的豚鼠中和抗体很低,但攻击感染后抗体迅速增长。第四天的抗体滴度为１：８～３２,第５天达１：１２８～２５６,第１４天抗体高达１：５１２～１０２４;而对照组抗体则上升很慢,第７天才出现低水平抗体（１：４）。血凝抑制抗体增长的动态与中和抗体近似。表明活疫苗免疫后虽然中和抗体水平不高,但一经感染可迅速产生高滴度抗体达到保护作用。     

Yellow fever/Japanese encephalitis chimeric viruses: construction and biological properties

A system has been developed for generating chimeric yellow fever/Japanese encephalitis (YF/JE) viruses from cDNA templates encoding the structural proteins prM and E of JE virus within the backbone of a molecular clone of the YF17D strain. Chimeric viruses incorporating the proteins of two JE strains, SA14-14-2 (human vaccine strain) and JE Nakayama (JE-N [virulent mouse brain-passaged strain]), were studied in cell culture and laboratory mice. The JE envelope protein (E) retained antigenic and biological properties when expressed with its prM protein together with the YF capsid; however, viable chimeric viruses incorporating the entire JE structural region (C-prM-E) could not be obtained. YF/JE(prM-E) chimeric viruses grew efficiently in cells of vertebrate or mosquito origin compared to the parental viruses. The YF/JE SA14-14-2 virus was unable to kill young adult mice by intracerebral challenge, even at doses of 10(6) PFU. In contrast, the YF/JE-N virus was neurovirulent, but the phenotype resembled parental YF virus rather than JE-N. Ten predicted amino acid differences distinguish the JE E proteins of the two chimeric viruses, therefore implicating one or more residues as virus-specific determinants of mouse neurovirulence in this chimeric system. This study indicates the feasibility of expressing protective antigens of JE virus in the context of a live, attenuated flavivirus vaccine strain (YF17D) and also establishes a genetic system for investigating the molecular basis for neurovirulence determinants encoded within the JE E protein.     

Chimeric yellow fever virus 17D-Japanese encephalitis virus vaccine: dose-response effectiveness and extended safety testing in rhesus monkeys

ChimeriVax-JE is a live, attenuated recombinant virus prepared by replacing the genes encoding two structural proteins (prM and E) of yellow fever 17D virus with the corresponding genes of an attenuated strain of Japanese encephalitis virus (JE), SA14-14-2 (T. J. Chambers et al., J. Virol. 73:3095-3101, 1999). Since the prM and E proteins contain antigens conferring protective humoral and cellular immunity, the immune response to vaccination is directed principally at JE. The prM-E genome sequence of the ChimeriVax-JE in diploid fetal rhesus lung cells (FRhL, a substrate acceptable for human vaccines) was identical to that of JE SA14-14-2 vaccine and differed from sequences of virulent wild-type strains (SA14 and Nakayama) at six amino acid residues in the envelope gene (E107, E138, E176, E279, E315, and E439). ChimeriVax-JE was fully attenuated for weaned mice inoculated by the intracerebral (i.c.) route, whereas commercial yellow fever 17D vaccine (YF-Vax) caused lethal encephalitis with a 50% lethal dose of 1.67 log(10) PFU. Groups of four rhesus monkeys were inoculated by the subcutaneous route with 2.0, 3.0, 4.0, and 5. 0 log(10) PFU of ChimeriVax-JE. All 16 monkeys developed low viremias (mean peak viremia, 1.7 to 2.1 log(10) PFU/ml; mean duration, 1.8 to 2.3 days). Neutralizing antibodies appeared between days 6 and 10; by day 30, neutralizing antibody responses were similar across dose groups. Neutralizing antibody titers to the homologous (vaccine) strain were higher than to the heterologous wild-type JE strains. All immunized monkeys and sham-immunized controls were challenged i.c. on day 54 with 5.2 log(10) PFU of wild-type JE. None of the immunized monkeys developed viremia or illness and had mild residual brain lesions, whereas controls developed viremia, clinical encephalitis, and severe histopathologic lesions. Immunized monkeys developed significant (>/=4-fold) increases in serum and cerebrospinal fluid neutralizing antibodies after i.c. challenge. In a standardized test for neurovirulence, ChimeriVax-JE and YF-Vax were compared in groups of 10 monkeys inoculated i.c. and analyzed histopathologically on day 30. Lesion scores in brains and spinal cord were significantly higher for monkeys inoculated with YF-Vax. ChimeriVax-JE meets preclinical safety and efficacy requirements for a human vaccine; it appears safer than yellow fever 17D vaccine but has a similar profile of immunogenicity and protective efficacy.     

不同年份生产的乙型脑炎减毒活疫苗SA14-14-2基因稳定性研究

为了研究不同年份生产的乙型脑炎减毒活疫苗病毒E蛋白基因稳定性,从分子水平控制乙型脑炎减毒活疫苗质量,确保疫苗安全性,本研究分析了不同年份生产的乙脑活疫苗病毒E蛋白基因核苷酸序列及编码的氨基酸序列,并与该疫苗原始种子、主种子、工作种子、乙脑病毒强、弱毒株进行比较。结果显示不同年份生产的乙脑活疫苗病毒E蛋白基因核苷酸序列与其原始种子、主种子、工作种子和基因库中登录的乙脑病毒弱毒株SA14-14-2的相应序列完全一致,与乙脑病毒强毒株SA14的E蛋白氨基酸序列比较有9个位点氨基酸发生了改变。不同年份生产的乙脑活疫苗病毒E蛋白基因稳定性表明该疫苗质量稳定、安全。     

动态浊度法检测冻干甲型肝炎减毒活疫苗细菌内毒素含量的研究

探讨动态浊度法检测冻干甲型肝炎减毒活疫苗细菌内毒素含量的可行性。参照《中国药典》2015年版通则1143细菌内毒素检测法中动态浊度法,对甲肝疫苗进行标准曲线可靠性试验、干扰初筛试验、干扰验证试验及内毒素含量的测定,同时与经凝胶法检定合格的同批疫苗进行比较。标准曲线可靠性试验结果符合规定。干扰初筛试验疫苗稀释160、320及640倍,回收率在50%~200%之间,均无干扰,符合要求。干扰验证试验结果进一步表明:疫苗稀释160倍对试验无干扰作用。采用动态浊度法检测的10批甲肝疫苗细菌内毒素含量均小于该疫苗的限值L=20 EU/m L,且与经凝胶法检定的同批疫苗结果一致。采用动态浊度法检测甲肝疫苗细菌内毒素含量是可行的,值得推广应用。     

Studies on live rubella vaccine. VI. Seroepidemiological surveillance and immunization of adolescents in high school.

The rubella seroimmunity status of a total of 1,204 students aged 12 to 19 in a junior and a senior high school in Osaka district was surveyed. Among these, 487 students (40.5%) were found to be seronegative (less than 1 : 8) by the hemagglutination inhibition (HI) test. A total of 287 students were immunized with live rubella vaccine, Biken lot No.7233. This caused an increased titer in all except one of the 262 seronegative students, while among 25 students with an initial HI antibody titer of 1 : 8 it caused more than 4-fold increase in 20 and 2-fold increase in 5. The vaccine caused no clinical manifestations, such as fever, rash or arthralgia.     

血凝抑制和病毒中和法在大流行流感疫苗临床血清学评价中的应用和比较

在大流行流感疫苗临床试验的免疫效果评价中,分别采用血凝抑制(HI)和病毒中和(VN)法检测血清中抗体滴度,并对结果进行比较。在中和试验中,分别以观察细胞病变法和血球凝集法确定中和法终点,两种方法得到的结果相近。376份血清(63.4%)以VN法和HI法检测获得结果相同。经统计学分析证明两种方法有很好的相关性,r值为0.85,HI 1∶40对应的VN滴度为1∶33。因此,在大流行流感疫苗免疫效果评价中,两种抗体检测方法有较好的一致性。