人用疫苗生产用细胞、胰酶及疫苗成品中猪细环病毒PCR检测方法的建立及验证  

Development and verification of a PCR assay for Torque teno sus virus in cells and trypsin for preparation of vaccine for human use as well as final product of the vaccine

作　　者：任芳芳[1] 钱兴丽[1] 宋彩花 陈巍[1] 赵勇 刘信毅 洪超[1] 杨晓蕾[1] REN Fang-fang;QIAN Xing-li;SONG Cai-hua;CHEN Wei;ZHAO Yong;LIU Xin-yi;HONG Chao;YANG Xiao-lei(Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming 650118, Yunnan Province, China)

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所,云南昆明650118

出　　处：《中国生物制品学杂志》2018年第6期656-661,共6页Chinese Journal of Biologicals

摘　　要：目的建立人用疫苗生产用细胞、胰酶及疫苗成品中猪细环病毒(Torque teno sus virus,TTSu V)的PCR检测方法,并进行验证。方法根据Gen Bank中登录的TTSu V1(JN688927)及TTSu V2(AY823991)的序列,合成TTSu V1及TTSu V2基因组部分DNA序列,连接于PUC57质粒上,制备重组质粒,以重组阳性质粒DNA为模板,PCR扩增316 bp的TTSu V1片段及252 bp的TTSu V2片段,同时验证方法的灵敏度和特异性。并采用该方法对2批0.1%胰酶、Vero、KMB17、MRC5、Hep2、BT细胞及6批疫苗[3批Sabin株脊髓灰质炎灭活疫苗(inactivated poliomyelitis vaccine made from Sabin strain,SIPV)收获液、1批SIPV成品、1批冻干甲型肝炎减毒活疫苗[hepatitis A(live)vaccine,freezedried,f HAV]成品、1批肠道病毒71型(enterovivus type 71,EV71)灭活疫苗成品]进行检测。结果建立的PCR法最低分别可检出10 fg/μL TTSu V1和0.01 fg/μL TTSu V2的目的基因;该方法可特异性扩增出TTSu V1和TTSu V2的目的基因条带。2批0.1%胰酶、Vero、KMB17、MRC5、Hep2、BT细胞及6批疫苗均未检出TTSu V1及TTSu V2。结论成功建立了疫苗生产用原材料、细胞、疫苗收获液及成品中TTSu V的PCR检测方法,且该方法具有较高的灵敏度和特异性,可用于本所疫苗生产中对TTSu V的检测。Objective To develop and verify a PCR assay for Torque teno sus virus type 1（TTSu V1） and type 2（TTSu V2） in the cells and trypsin for production of vaccine for human use as well as the final product of vaccine.Methods Partial DNA sequences of TTSu V1（JN688927）and TTSu V2（AY823991）genomes were synthesized according to the two sequences in Gen Bank,and inserted into vector p UC57. The constructed recombinant plasmids were used as templates for TTSu V1 gene fragment at a length of 316 bp and TTSu V2 gene fragment at a length of 252 bp by PCR. The PCR assay was verified for sensitivity and specificity,by which Vero,KMB17,MRC5,Hep2 and BT cells,two batches of 0. 1% trypsin,three batches of virus harvests of inactivated poliomyelitis vaccine made from Sabin strain（SIPV）,one batch of final product of SIPV,one batch of final product of hepatitis A（live） vaccine,freeze-dried（f HAV）,and one batch of final product of inactivated enterovirus type 71 vaccine（EV71） were tested. Results The minimum detection limits of developed PCR assay for TTSu V1 and TTSu V2 were 10 and 0. 01 fg/μL respectively. Specific TTSu V1 and TTSu V2 genes were amplified by the developed PCR assay. No TTSu V1 and TTSu V2 DNAs were found in two batches of trypsin,Vero,KMB17,MRC5,Hep2 and BT cells as well as six batches of vaccines. Conclusion A PCR assay for TTSu V raw materials and cells for vaccine production as well as harvest and final product of vaccine was successfully developed,which showed high sensitivity and specificity,and might be used for the determination of TTSu V in vaccine production in Institute of Medical Biology,Chinese Academy of Medical Science and Peking Union Medical College.

关 键 词：猪细环病毒 细胞 胰酶 减毒活疫苗 灭活疫苗 聚合酶链式反应 

分 类 号：Q789[生物学—分子生物学] R392-33[医药卫生—免疫学]