目的:本文以对生物制品生物效价检测进行规范的《中国药典》2020年版新增9401指导原则为标准进行EV71抗原检测方法的验证,进一步浅析生物制品生物活性/效价检测的方法学验证.方法:依据《中国药典》2020年版9401指导原则,结合EV71抗原检测方法特点及验证目的对其进行专属性、相对准确度、精密度、线性和范围5个方面进行方法学验证.结果:EV71抗原检测方法专属性强,6个效价水平几何变异系数为4.7％-13.0％,均小于15％,相对偏倚为-6.05％～9.03％,均在±15％范围内,定量范围为225％～31％,线性回归相关系数为0.999 5,5方面验证均符合要求,且适用于该检测方法的使用.结论:本原则可具体指导生物学检测方法验证,在对不同检测方法进行验证时,根据检测目的、方法的原理、方法的技术特点、被检物的成分等设计具体方案.

目的 探讨mircoRNA基因多态性与云南汉族人群非小细胞肺癌发生发展的相关性.方法 选取云南地区汉族人群非小细胞肺癌患者415例,健康对照460例,采用Taqman探针基因分型法对3个SNP位点rs2292832 T＞C(pre-miR-149)、rs107822 C＞T(pre-miR-219a-1),rs629367 C＞A(pri-let-7a-1)进行基因分型并分析其等位基因、基因型在非小细胞肺癌患者(按病理分层为鳞癌、腺癌及其他类型肺癌;按临床分期分层为Ⅰ+Ⅱ期、Ⅲ+Ⅳ期)及健康对照中的频率差异.结果rs2292832、rs107822、rs629367位点的等位基因、基因型频率在非小细胞肺癌组和对照组间的差异没有统计学意义,在分层分析及遗传模式分析中也无显著性差异.结论rs2292832、rs107822、rs629367可能与云南汉族人群非小细胞肺癌无相关性.

目的 比较干燥保存及湿保存肠道病毒71型(enterovirus 71,EV71)抗原检测板的稳定性.方法 用羊抗EV71多克隆抗体包被96孔酶标板,制备20块EV71抗原检测板,10块板置空气干燥后真空包装,-25℃保存;另外10块板用洗涤液浸泡,封口后置4℃保存.分别于保存0、20、40、60、90、120、180d时,检测5份EV71样品.结果 干燥保存EV71抗原检测板检测5份样品7个时间点的均值分别为315.0、299.4、7 795.3、827.6、261.4 U/mL,湿保存的EV71抗原检测板检测结果均值分别为354.3、347.1、6 766.6、749.7、277.3 U/mL,各时间点两种方法检测结果差异无统计学意义(P＞0.05).两种方法保存180 d时,EV71抗原检测板均可保持较好的活性(P＞0.05).结论 两种方法保存EV71抗原检测板6个月内,均具有良好的稳定性.

目的 对Sabin株脊髓灰质炎灭活疫苗(Sabin inactivated poliovirus vaccine,sIPV)在生产过程中的灭活效果进行验证及评价.方法 分别制备Sabin株Ⅰ、Ⅱ及Ⅲ型脊髓灰质炎病毒连续5批纯化液,加入终浓度为92.5 μg/mL的甲醛,37℃灭活12d.收集灭活前(0 d)及灭活1～6、9和12 d样品,采用人喉癌上皮细胞(Hep-2)通过细胞病变法检测0～6 d样品的病毒滴度,ELISA法检测不同灭活时间样品的D抗原含量,并验证9及12d样品的灭活效果.结果 Sabin株Ⅰ、Ⅱ及Ⅲ型脊髓灰质炎病毒均在灭活第3天下降至0.00 lgCCID50/mL,3种型别病毒同日的灭活速率差异无统计学意义(P＞0.05).灭活动力学曲线显示,0～3d样品的病毒滴度呈直线下降,病毒灭活效果与灭活时间密切相关(Ⅰ、Ⅱ及Ⅲ型相关系数r分别为-0.995、-0.976及-0.996,R2分别为0.991、0.952及0.992).Ⅰ、Ⅱ及Ⅲ型病毒12d样品D抗原平均回收率分别为80％、85％和83％,抗原有一定的损失,但差异无统计学意义(P＞0.05).灭活9及12 d样品灭活验证结果均为阴性.结论 Sabin株脊髓灰质炎病毒在目前的灭活工艺条件下,均可稳定地被灭活,且能收获含量较高的D抗原,保证了sIPV疫苗的稳定性及安全性.

目的 鉴定本所自主建立的人用疫苗生产用人二倍体细胞株KMB17的正确性.方法 用染色体核型检查法和短串联重复序列(short tandem repeat,STR)基因分型法对本所疫苗生产用人二倍体细胞株KMB17进行鉴定.将疫苗生产用KMB17细胞经秋水仙素处理,积累中期相染色体后,低渗条件下释放出染色体,吉姆萨染料染色制片,镜检,观察染色体结构,精确计数100个细胞的染色体数目;选取人源细胞的20个STR位点对KMB17细胞进行DNA分型,并将获得的STR图谱与ATCC和DSMZ数据库进行比对分析.结果 高倍镜下观察可见,样品细胞染色体组结构正常,无缺失和突变;精确计数100个细胞中,染色体数为46的细胞数有84个,无染色单体和染色体断裂细胞,无结构异常细胞,亚二倍体细胞14个,超二倍体细胞数2个.STR基因分型获得的特征性图谱清晰,未出现三单位基因,在ATCC和DSMZ数据库中均未找到与其细胞分型完全匹配的细胞株.结论 本所自主建立的疫苗生产用人二倍体细胞株KMB17的细胞染色体结构和数目均正常,不存在污染和交叉污染,为正确细胞株.

目的 建立人用疫苗及生产用细胞中猪圆环病毒1型(porcine circovirus 1,PCV1)和2型(PCV2)PCR检测方法,并进行验证.方法 参照GenBank中登录的PCV1(KC447455)和PCV2(AY578327)序列,全基因组合成PCV1和PCV2基因序列,以合成的PCV1和PCV2全基因组序列DNA为模板,进行PCR扩增,对PCR反应中的退火温度进行优化.对优化后的PCR方法进行灵敏度和特异性验证,并用该方法检测疫苗生产用细胞KMB17、Vero工作种子批、脊髓灰质炎减毒活疫苗(oral poliomyelitis vaccine,OPV)收获液、冻干甲型肝炎减毒活疫苗(freeze-dried hepatitis A vaccine,fHAV)成品、肠道病毒71型(enterovirus 71,EV71)灭活疫苗收获液及成品、Sabin株脊髓灰质炎灭活疫苗(inactivated poliomyelitis vaccine prepared with Sabin strain,SIPV)收获液及成品的PCV1和PCV2.结果 优化后的退火温度为63℃,建立的PCR方法最低可检测出10-1 fg/μL的目的基因,仅对PCV基因组DNA能扩增出特异性条带.用该方法检测生产用细胞KMB17及Vero工作种子批、OPV收获液、fHAV成品、EV71灭活疫苗收获液及成品、SIPV收获液及成品,均未检出PCV1和PCV2.结论 成功建立了生产用细胞种子、疫苗收获液及成品中PCV的PCR检测方法,该方法具有较高的灵敏度和较强的特异性,能够用于本所生物制品生产用细胞种子、疫苗收获液及成品中PCV污染的检测,从而提高生物制品的安全性.

Objective To evaluate the recovery of polio virus Sabin strain of three types after dialysis.Methods Polio virus Sabin strain of types Ⅰ,Ⅱ and Ⅲ,at high titers,were dialyzed.The recoveries of titers of each type before and after dialysis,as well as the clearance rates of formaldehyde after dialysis,were compared.However,the polio virus at titers of 101,100 and 10-1 CCID50/mL,and the samples of the same type and titer before and after dialysis were inoculated to Hep-2 cells and subcultured for three passages.The cytopathic effect (CPE) rates and virus titers before and after dialysis were compared.Results The volume and titer of samples of various types at high titers after dialysis showed no significant difference with those before dialysis (P ＞ 0.05),while the clearance rate of formaldehyde after dialysis was more than 99％.All the samples of various types at a titer of 101 CCID50/mL caused CPE after subculture for three passages.The CPE rates caused by samples of types Ⅰ,Ⅱ and Ⅲ,each at a titer of 100 CCID50/mL,were 66％,33％and 33％ respectively after subculture for one passage,all of which were 100％ after subculture for two and three passages.However,the samples of various types at a titer of 10-1 CCID50/mL caused no CPE after subculture for three passages.All the virus titers of samples after CPE were more than 6.0 lgCCID50/mL.Conclusion The recovery of three types of polio virus Sabin strain of three types were high after dialysis,with little loss,indicating that dialysis might be used for purification of the virus.

Objective To authenticate the working cell bank for production of vaccines for human use by two methods,and provide a guarantee for improving the accuracy of cell lines for production of vaccines for human use and the safety of vaccines.Methods Short tandem repeat (STR) markers were used to sequence the eight loci,i.e.D8Sl106,D1S518,D6S1017,D17S1304,D4S2408,D5S1467,D19S245 and DYS389,in Vero cell lines,and the profiles were compared with those reported in documents.The prepared Vero cell lines for vaccine production were treated with stained with Giemsa staining,and the chromosomes of 100 cells were counted precisely under microscope,based on which the proportion of cells with 58 or 60 chromosomes was calculated.Results The profiles of the Vero cell lines are identical to those reported in documents.No three unit genes appeared,and the numbers of repeats were identical The proportion of cells with 58 or 60 chromosomes was 79％.Conclusion The Vero cell lines from the working cell bank for vaccine production were correct cell lines without contamination or cross-contamination,which provided a guarantee for the safety and accuracy of produced vaccines for human use.