论文部分内容阅读
建立重组复制型溶瘤腺病毒p53(SG600-P53)的质控检测方法与质量标准。采用限制性内切酶酶切、PCR法对端粒酶启动子、低压缺氧调控元件融合的巨细胞病毒(cytomegalovirus,CMV)启动子、p53基因等重组病毒载体结构进行分析,鉴定结果均与理论值相符。经紫外吸收法(A260)检测,病毒颗粒数为2.0×1011 VP.mL-1;TCID50法测定感染活性为5.0×1010 IU.mL-1。p53蛋白ELISA检测结果表明,重组病毒体外感染人肺癌细胞H1299后,感染组核蛋白和空白对照组A450吸收度之比为5.2。该基因治疗制剂对人肺癌细胞A549体外杀伤的MOIIC50为1.0。以相同MOI感染并经TCID50法检测,重组病毒在人肺癌细胞A549与人表皮成纤维二倍体细胞BJ的增殖比值为398。经阴离子高效液相色谱分析,病毒载体颗粒纯度为99.5%。定量PCR检测表明在1×107 VP的病毒颗粒中,野生型腺病毒基因片段少于1个拷贝。本研究建立的重组复制型溶瘤腺病毒的质量标准与检测方法,可用于该制品的质量控制,同时也为其他溶瘤基因治疗病毒载体质控研究提供参考。
To establish a quality control test method and quality standard for recombinant replicating oncolytic adenovirus p53 (SG600-P53). The structure of recombinant viral vector such as cytomegalovirus (CMV) promoter and p53 gene fused with the promoter of telomerase and hypobaric hypoxia regulatory element was analyzed by restriction endonuclease digestion and PCR. The identification results were in agreement with The theoretical value is consistent. The number of virus particles was 2.0 × 10 11 VP.mL-1 by ultraviolet absorption method (A260), and the infection activity was 5.0 × 1010 IU · mL-1 by TCID50 method. The results of p53 protein ELISA showed that the ratio of A450 absorbance of the infected group to that of the blank control group was 5.2 after the recombinant virus was infected in vitro by human lung cancer cell line H1299. The MOI IC50 of the gene therapy preparation against human lung cancer cell A549 in vitro was 1.0. With the same MOI infection and TCID50 assay, the proliferation ratio of recombinant virus in human lung cancer cell A549 and human epidermal fibroblasts diploid cells BJ was 398. By anion-high performance liquid chromatography, the purity of the viral vector particles was 99.5%. Quantitative PCR assays showed that less than 1 copy of the wild-type adenovirus gene fragment was found in 1 × 10 7 VP virions. The quality standard and test method of recombinant replicated oncolytic adenovirus established in this study can be used for the quality control of the product and also provide reference for other oncolytic gene therapy viral vector quality control research.