Q Sepharose Fast Flow(Q Sepharose F.F.) was adopted to purify the suspensions of type Ⅰ,Ⅱ,Ⅲ oral poliovaccine (OPV), which is prepared from Vero cells. After clarification and concentration by ultrafiltration,th...Q Sepharose Fast Flow(Q Sepharose F.F.) was adopted to purify the suspensions of type Ⅰ,Ⅱ,Ⅲ oral poliovaccine (OPV), which is prepared from Vero cells. After clarification and concentration by ultrafiltration,the recovery of virus infected titre may attain above 85%.Using column chromatography on Q Sepharose F.F. to purify the concentrated virus suspensions,the purified viruses attain 100% recovery of virus infectivity. Dot membrane hybrization was used to detect DNA with the probe of Vero cell genome DNA which was labeled with α P 32 dATP,the contents of residual substrate DNA was less than 100 Pg/dose. The process of downstream had no significant influence on some biological characters of purified viruses,such as virus morphology ,tumorigenicity, rct/40 character and capsid protein.This downstream reseach indicates that Q Sepharose F.F. is an ideal chromatography material for purifying OPV prepared from Vero cells.展开更多
目的克隆Vero细胞基因组DNA的长串联重复序列(Long tandem repeats,LTR),并建立Vero细胞DNA定量PCR检测方法。方法提取Vero细胞基因组DNA,经HindⅢ酶切后,克隆至pET-30a(+)载体上,酶切及测序鉴定,并与Gen-Bank中登录的序列进行同源性比...目的克隆Vero细胞基因组DNA的长串联重复序列(Long tandem repeats,LTR),并建立Vero细胞DNA定量PCR检测方法。方法提取Vero细胞基因组DNA,经HindⅢ酶切后,克隆至pET-30a(+)载体上,酶切及测序鉴定,并与Gen-Bank中登录的序列进行同源性比较。以172 bp序列为靶基因设计定量PCR引物和Taq探针,建立Vero细胞DNA定量PCR检测方法,并进行特异性验证。结果经酶切及测序鉴定,共获得8个阳性克隆质粒pET-30a-172,与GenBank中登录的基因序列同源性在98.3%～99.4%之间;建立的Vero细胞DNA定量PCR检测方法的灵敏度可达1×10-6 ng/μl,线性范围为1 ng/μl～1×10-6 ng/μl;以建立的方法检测鸡胚细胞、CHO细胞和地鼠肾细胞DNA,均未见扩增曲线,仅人二倍体细胞(MRC-5)DNA有明显的扩增曲线,表明该法特异性良好。结论已成功建立了以172 bp LTR为靶基因的Vero细胞DNA定量PCR检测方法,可用于疫苗中Vero细胞DNA残留量的检测。展开更多
目的对Taq Man MGB探针实时定量PCR检测Vero细胞残余DNA的方法进行适用性验证及应用。方法对Taq Man MGB探针实时定量PCR检测Vero细胞残余DNA的方法进行特异性、灵敏度、精密性、准确性验证,并应用该方法检测3批次脊髓灰质炎病毒浓缩液...目的对Taq Man MGB探针实时定量PCR检测Vero细胞残余DNA的方法进行适用性验证及应用。方法对Taq Man MGB探针实时定量PCR检测Vero细胞残余DNA的方法进行特异性、灵敏度、精密性、准确性验证,并应用该方法检测3批次脊髓灰质炎病毒浓缩液、纯化液和原液中Vero细胞残余DNA含量,计算Vero细胞残余DNA去除率。结果 Taq Man MGB探针实时定量PCR能够特异性扩增Vero细胞基因组DNA长串连重复序列;DNA浓度在10-1~10-6 ng/μl范围内标准曲线线性良好,相关系数为0.996,扩增效率为93.54%;检测灵敏度为10-6 ng/μl,高于斑点杂交法;检测高（10-2 ng/μl）、中（10-4 ng/μl）、低（10-6 ng/μl）浓度阳性对照DNA模板的试验内变异系数（CV）为0.145%~3.110%,试验间CV为0.624%~2.359%;检测不同浓度阳性对照DNA的回收率在101.5%~109.4%之间,均在定量方法可接受的回收率范围内;在斑点杂交检测灵敏度范围内,同一样品采用Taq Man MGB探针实时定量PCR法与斑点杂交法半定量检测的结果吻合。3批次脊髓灰质炎病毒浓缩液纯化后可有效去除Vero细胞残余DNA,总去除率约为100%。结论 Taq Man探针实时定量PCR法特异性、精密性、准确性良好,灵敏度高,可作为斑点杂交法的替代方法,用于实验室内部的质量控制。展开更多
文摘Q Sepharose Fast Flow(Q Sepharose F.F.) was adopted to purify the suspensions of type Ⅰ,Ⅱ,Ⅲ oral poliovaccine (OPV), which is prepared from Vero cells. After clarification and concentration by ultrafiltration,the recovery of virus infected titre may attain above 85%.Using column chromatography on Q Sepharose F.F. to purify the concentrated virus suspensions,the purified viruses attain 100% recovery of virus infectivity. Dot membrane hybrization was used to detect DNA with the probe of Vero cell genome DNA which was labeled with α P 32 dATP,the contents of residual substrate DNA was less than 100 Pg/dose. The process of downstream had no significant influence on some biological characters of purified viruses,such as virus morphology ,tumorigenicity, rct/40 character and capsid protein.This downstream reseach indicates that Q Sepharose F.F. is an ideal chromatography material for purifying OPV prepared from Vero cells.
文摘目的克隆Vero细胞基因组DNA的长串联重复序列(Long tandem repeats,LTR),并建立Vero细胞DNA定量PCR检测方法。方法提取Vero细胞基因组DNA,经HindⅢ酶切后,克隆至pET-30a(+)载体上,酶切及测序鉴定,并与Gen-Bank中登录的序列进行同源性比较。以172 bp序列为靶基因设计定量PCR引物和Taq探针,建立Vero细胞DNA定量PCR检测方法,并进行特异性验证。结果经酶切及测序鉴定,共获得8个阳性克隆质粒pET-30a-172,与GenBank中登录的基因序列同源性在98.3%～99.4%之间;建立的Vero细胞DNA定量PCR检测方法的灵敏度可达1×10-6 ng/μl,线性范围为1 ng/μl～1×10-6 ng/μl;以建立的方法检测鸡胚细胞、CHO细胞和地鼠肾细胞DNA,均未见扩增曲线,仅人二倍体细胞(MRC-5)DNA有明显的扩增曲线,表明该法特异性良好。结论已成功建立了以172 bp LTR为靶基因的Vero细胞DNA定量PCR检测方法,可用于疫苗中Vero细胞DNA残留量的检测。
文摘目的对Taq Man MGB探针实时定量PCR检测Vero细胞残余DNA的方法进行适用性验证及应用。方法对Taq Man MGB探针实时定量PCR检测Vero细胞残余DNA的方法进行特异性、灵敏度、精密性、准确性验证,并应用该方法检测3批次脊髓灰质炎病毒浓缩液、纯化液和原液中Vero细胞残余DNA含量,计算Vero细胞残余DNA去除率。结果 Taq Man MGB探针实时定量PCR能够特异性扩增Vero细胞基因组DNA长串连重复序列;DNA浓度在10-1~10-6 ng/μl范围内标准曲线线性良好,相关系数为0.996,扩增效率为93.54%;检测灵敏度为10-6 ng/μl,高于斑点杂交法;检测高（10-2 ng/μl）、中（10-4 ng/μl）、低（10-6 ng/μl）浓度阳性对照DNA模板的试验内变异系数（CV）为0.145%~3.110%,试验间CV为0.624%~2.359%;检测不同浓度阳性对照DNA的回收率在101.5%~109.4%之间,均在定量方法可接受的回收率范围内;在斑点杂交检测灵敏度范围内,同一样品采用Taq Man MGB探针实时定量PCR法与斑点杂交法半定量检测的结果吻合。3批次脊髓灰质炎病毒浓缩液纯化后可有效去除Vero细胞残余DNA,总去除率约为100%。结论 Taq Man探针实时定量PCR法特异性、精密性、准确性良好,灵敏度高,可作为斑点杂交法的替代方法,用于实验室内部的质量控制。
