牛痘病毒粒细胞巨噬细胞集落刺激因子注射液的生物学活性测定方法研究

目的： 建立牛痘病毒粒细胞巨噬细胞集落刺激因子（vaccinia virus/GM-CSF）注射液的生物学活性测定方法。方法： 采用噬斑法进行感染滴度测定，结合定量聚合酶链反应（quantitative polymerase chain reaction，Q-PCR）进行病毒基因组滴度/感染滴度测定；采用TF-1细胞增殖法，四参数方程拟合处理试验数据，检测不同浓度梯度的样品感染Hela细胞后表达的GM-CSF生物学活性；采用ELISA法，二次方程拟合处理试验数据，检测不同浓度梯度样品感染Hela细胞后的GM-CSF的表达量；采用ELISA法进行β半乳糖苷酶活性测定；采用肿瘤细胞（UACC 257）和正常细胞（GM 00038）进行溶瘤活性测定。结果： 测定了1批牛痘病毒粒细胞巨噬细胞集落刺激因子注射液，其病毒感染滴度为6.5×10⁸ PFU·mL^-1，病毒基因组滴度/感染滴度为27，各浓度梯度孔GM-CSF表达量均大于10 pg，GM-CSF生物学活性为181 rGCU，溶瘤活性为144 rOnU，β-半乳糖苷酶活性为124 rBU。结论： 对牛痘病毒GM-CSF注射液的生物学活性进行了相对全面的测定，各个项目均符合规定，能够用于该产品的质量控制，同时对同类产品的质量控制具有借鉴意义。

Objective: To establish methods for biological activity determination of vaccinia virus/granulocyte macrophage colony stimulating factor(GM-CSF)injection.Methods: Infectious titer was measured by plaque assay and viral genome titer/infectious titer was measured by a combination of quantitative polymerase chain reaction (Q-PCR)and plaque assay.The biological activity of GM-CSF was detected by TF-1 cell proliferation assay and curve fitting was performed by four-parameter equation.The expression of GM-CSF in Hela cells infected by different concentrations of virus was detected by ELISA method and curve fitting was performed by quadratic equation.The activity of β-galactosidase was determined by ELISA. The oncolytic activity was determined by the tumor cells(UACC 257) and normal cells(GM 00038).Results: A batch of vaccinia virus/GM-CSF injection was determined.The viral infectious titer was 6.5×10⁸ PFU·mL^-1,viral genome titer/infectious titer was 27,GM-CSF expression level was more than 10 pg in all gradient wells,biological activity of GM-CSF was 181 rGCU,oncolytic activity was 144 rOnU,and β-galactosidase activity was 124 rBU. Conclusion: The biological activity of vaccinia virus/GM-CSF injection has been determined in a relatively comprehensive way,and all the items meet the requirements,which can be used for the quality control of related products,and also provide significance reference for the quality control of similar products.

[1] RUSSELL L,PENG KW.The emerging role of oncolytic virus therapy against cancer[J].Chin Clin Oncol,2018,7(2):16
[2] KAUFMAN HL,KOHLHAPP FJ,ZLOZA A.Oncolytic viruses:a new class of immunotherapy drugs[J].Nat Rev Drug Discov,2015,14(9):642
[3] NEIDEL S,REN H,TORRES AA,et al.NF-κB activation is a turn on for vaccinia virus phosphoprotein A49 to turn off NF-κB activation[J].Proc Natl Acad Sci USA,2019,116(12):5699
[4] DENG L,FAN J,DING Y,et al.Oncolytic cancer therapy with a vaccinia virus strain[J].Oncol Rep,2019,41(1):686
[5] BENDJAMA K,QUEMENEUR E.Modified Vaccinia virus Ankara-based vaccines in the era of personalized immunotherapy of cancer[J].Hum Vaccin Immunother,2017,13(9):1997
[6] 于雷,李永红,秦玺,等.Q-PCR法检测腺病毒基因治疗产品中的复制性腺病毒[J].生物技术通讯,2017,28(3):352 YU L,LI YH,QIN X,et al.Detection of replication-competent adenoviruses in adenoviral gene therapy products by Q-PCR[J].Lett Biotechnol,2017,28(3):352
[7] 秦玺,李永红,杨琦,等.实时荧光定量PCR法用于重组SIV-hPEDF注射液病毒颗粒数的检测[J].现代生物医学进展,2015,15(8):1401 QIN X,LI YH,YANG Q,et al.Quantitation of virus particles in recombinant SIV-hPEGF injection by real-time PCR[J].Prog Mod Biomed,2015,15(8):1401
[8] 毕华,李永红,韩春梅,等.牛痘病毒GM-CSF注射液中GM-CSF活性及表达量的检测[J].中国生物制品学杂志,2018,31(9):1015 BI H,LI YH,HAN CM,et al.Determination of activity and expression level of GM-CSF in vaccinia virus/GM-CSF injection[J].Chin J Biol,2018,31(9):1015
[9] 李永红,毕华,史新昌,等.人用基因治疗制品生产和质量控制的通用性技术要求[J].中国新药杂志,2018,27(21):2482 LI YH,BI H,SHI XC,et al.General technical requirements for production and quality control of human gene therapy products[J].Chin J New Drugs,2018,27(21):2482
[10] 李春辉,胡泊,翁郁华,等.基因治疗的现状与临床研究进展[J].生命科学仪器,2019,17(8):3 LI CH,HU B,WENG YH,et al.Current situation and clinical research progresses of gene therapy[J].Life Sci Instrum,2019,17(8):3