摘要
目的:建立相对荧光实时定量法(RT-QPCR 2_(-ΔΔCt)),考察聚乙二醇化重组人干扰素α2b注射液临床Ⅰ期试验中给药后外周全血中2',5'-寡聚腺苷酸合成酶(2',5'-OAS)相对药前点的相对表达水平变化。方法:根据Gen Bank中查询的2',5'-OAS的序列构建质粒,并纯化定量作为方法标准品,制作标准曲线及验证样品,考察该方法的准确度及精密度;采用RT-QPCR 2_(-ΔΔCt)方法,以个体药前样品为对照,GAPDH为内参,比较考察给予聚乙二醇化重组人干扰素α2b注射液(受试品160μg/人)与阳性对照药派罗欣(180μg/人)后外周血中2',5'-OAS mRNA表达变化倍数;同时,基于ELISA方法对2',5'-OAS蛋白浓度表达进行分析。结果:2',5'-OAS标准曲线有相对较好的线性(R2=0.99),cDNA 2',5'-OAS与2',5'-OAS标准品的PCR产物熔解曲线温度一致,说明引物特异性好。2个剂量组中m RNA表达水平均在20 h左右达到峰值,其中160μg剂量组中峰浓度较高。ELISA检测血清中2',5'-OAS浓度无明显变化。结论:与传统蛋白水平检测的ELISA方法相比,RT-QPCR 2_(-ΔΔCt)灵敏度较高,可以作为一种检测外周全血中生物标志物的替代方法,以给予药效学研究的临床支持。
Objective: To develop a relative quantitative RT-QPCR method to investigate the expression of 2',5'-oligoadenylate synthase(2',5'-OAS) in the peripheral blood in clinical phaseⅠtrial. Methods: The plasmids were constructed according to the sequence of 2',5'-OAS in Gen Bank, and the standard and quality control samples were prepared using plasmid. The accuracy and precision of the method were investigated. GAPDH was used as the control and RT-QPCR 2^(-ΔΔCt)method. In clinicalⅠtrial, one group was treated with PEGylated recombinant human interferon α2b injection(160 μg/person) and positive control peginterferon(180 μg/person). Results were compared from the 2',5'-OAS m RNA and protein expressionbased on RT-QPCR and ELISA. Results: 2',5'-OAS standard curve has a relatively good linearity(R2=0.99). Consistent melting curve temperature derived from cDNA template and standards indicated good primer specificity. The m RNA expression levels in the two dose groups reached peak at about 20 h, and the peak concentration was higher in the 160 μg dose group. There was no significant change in the 2',5'-OAS concentration in serum using ELISA. Conclusion: Compared with the traditional ELISA method, RT-QPCR 2-ΔΔCthas superior performance due to high sensitivity, and it can be used as a alternative method for detection of biomarkersin peripheral blood in clinical pharmacodynamics study.
出处
《生物技术通讯》
CAS
2018年第1期78-83,共6页
Letters in Biotechnology
基金
国家重大专项(2015ZX09501007
2015ZX09501008)
作者简介
张晶（1988-）,女,硕士研究生,（E-mail）kristinezhang@up-pharma.com;通信作者：付洁,（E-mail）13311161618@163.com;;宋海峰,（E-mail）bapklab@yahoo.com
