rAAV2-ND4注射液基因转录水平的生物学活性检测方法

Biological activity detection method for gene transcription level of rAAV2-ND4 injection

目的:建立并验证检测rAAV2-ND4注射液转录水平的生物学活性检测方法。方法:rAAV2-ND4样品(3.0×10^(10)vg·mL^(-1),以每孔360μL)感染HEK293T细胞(3.0×105cells·mL^(-1),每孔0.8 mL,37℃5%二氧化碳培养预培养24 h)后2 h,加适量培养基继续培养24 h,收集细胞;按细胞总RNA提取试剂盒操作细则提取细胞总RNA(裂解细胞、吸附、去蛋白、去杂质和洗脱);将提取后的细胞总RNA为模板,用qPCR方法分别检测ND4的mRNA Ct值和β-actin内参基因mRNA Ct值。通过同时设立rAAV2-ND4参比品进行平行检测,通过β-actin内参基因和rAAV2-ND4参比品双重校正检测rAAV2-ND4相对于其参比品的生物学活性。对所建立方法进行方法学验证,包括准确性、精密度、线性和专属性等。另考察了3批制品的批间一致性。结果:qPCR检测ND4 mRNA转录量和参比品校正的方法检测结果提示ND4 mRNA与rAAV2-ND4感染细胞滴度之间存在量效关系。经方法学验证,方法的检测值与预期值之间偏离率在100%~130%;不同时间不同人员的9次结果RSD为13.75%;rAAV2-ND4滴度在1.0×10^(10)~5.0×10^(10)vg·mL^(-1)范围内方法线性r>0.97;方法具有很好的专属性。3批rAAV-ND4检测值的RSD为9.3%。结论:初步建立了rAAV2-ND4转录水平的生物学活性检测方法。

Objective:To establish and validate a biological activity detection method for detecting the transcription level of rAAV2-ND4 injection.Methods:rAAV2-ND4 sample(3.0×10^(10)vg·mL^(-1)at 360μL per well)was infected with HEK293T cells(3.0×10^(5) cells·mL^(-1),0.8 mL per well,pre-cultured at 37℃with 5%carbon dioxide for 24 h),followed by 2 h of culture.An appropriate amount of culture medium was added and continued to culture for 24 h,then the cells were collected.Cell total RNA was extracted according to the operating instructions of the cell total RNA extraction kit(cell lysis,adsorption,deproteinization,impurity removal,and elution).Using the extracted total RNA of cells as a template,the Ct values of mRNA andβ-actin internal reference gene of ND4 were detected using qPCR method.By simultaneously setting up rAAV2-ND4 reference samples for parallel testing,the relative biological activity of rAAV2-ND4 was detected using double calibration detection withβ-actin internal reference genes and the rAAV2-ND4 reference substance.The methodological validation included accuracy,precision,linearity,and specificity.The inter batch consistency of the three products was also investigated.Results:The results of qPCR detection of ND4 mRNA transcription and reference correction method indicated a dose-response relationship between ND4 mRNA and rAAV2 ND4 infect titers.According to the verification of methodology,the deviation rate between the detection value and the expected value of the method was between 100%and 130%.The RSD of 9 results for different personnel at different times was 13.75%.The method was linearity from 1.0×10^(10)to 5.0×10^(10)vg·mL^(-1)of rAAV2-ND4 titer,r>0.97.The method had good specificity.The RSD of the three batches of rAAV-ND4 detection values was 9.3%.Conclusion:A preliminary biological activity detection method for rAAV2 ND4 transcription level has been established.