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目的研究建立重组复制型溶瘤单纯疱疹病毒人细胞巨噬细胞集落刺激因子(GM-CSF,HSV1/GM-CSF)的质控方法与质量标准。方法采用限制性内切酶酶切与PCR法,对GM-CSF基因、单纯疱疹病毒载体ICP 34.5、ICP6、ICP47等改造结构进行鉴定。采用A260紫外吸收法检测病毒颗粒数;pfu法测定HSV1/GM-CSF感染活性。HSV1/GM-CSF体外感染Vero细胞后,分别测定感染上清中GM-CSF表达量以及表达产物的生物学活性;测定HSV1/GM-CSF对肿瘤细胞MCF7的体外杀伤活性;HSV1/GM-CSF分别以相同MOI感染MCF7与二倍体人胚肺成纤维细胞MRC5后,分别测定细胞裂解上清中子代病毒的感染活性,并以子代病毒的感染活性比值来分析该制品在肿瘤细胞中的增殖复制能力。采用PCR法控制野生型单纯疱疹病毒等外源因子的残留。结果对重组HSV1/GM-CSG基因组的酶切、以及对所携带的GM-CSF基因、ICP 34.5、ICP6、ICP47基因改造区的PCR鉴定结果与理论值相符。病毒载体颗粒数为4.5×1010VP.mL-1,感染活性为4.3×107pfu.mL-1。HSV1/GM-CSF以MOI 0.01感染Vero细胞72 h后,ELISA检测上清中GM-CSF表达量为228 ng.mL-1,表达产物的生物学活为4.3×103U.mL-1。该基因治疗制剂对人乳腺癌肿瘤细胞MCF7体外杀伤的MOIIC50为0.08。以相同MOI感染并经pfu法检测,HSV1/GM-CSF在人乳腺癌肿瘤细胞MCF7与人胚肺成纤维二倍体细胞MRC5的增殖比值为308。未检出野生型单纯疱疹病毒。其他各项指标均符合《人基因治疗研究和制剂质量控制技术指导原则》及2005年版《中国药典》(三部)要求。结论初步建立了HSV1/GM-CSF的质控方法和质量标准,并已用于该制品的质量控制。
Objective To study the quality control method and quality standard of recombinant human oncolytic herpes simplex virus (GM-CSF) human macrophage colony-stimulating factor (GMV-CSF, HSV1 / GM-CSF) Methods The recombinant plasmids of GM-CSF gene, herpes simplex virus (ICP) 34.5, ICP6 and ICP47 were identified by restriction endonuclease digestion and PCR. A260 UV absorption method was used to detect the number of virus particles; pfu method to determine HSV1 / GM-CSF infection activity. After in vitro infection of Vero cells with HSV1 / GM-CSF, the expression of GM-CSF in the supernatant of the transfected HSV1 / GM-CSF and the biological activity of the expressed product were assayed. The in vitro cytotoxic activity of HSV1 / GM- After infecting MCF7 and diploid human embryo lung fibroblast MRC5 at the same MOI respectively, the infectious activity of the progeny virus in the cell lysate supernatant was measured, and the ratio of the progeny virus to the infectivity of the progeny virus was used to analyze the product in the tumor cells Proliferative and replication ability. PCR method to control wild-type herpes simplex virus and other exogenous factors. Results The results of PCR identification of the recombinant HSV1 / GM-CSG genome and the GM-CSF gene carried by the recombinant plasmids of ICP 34.5, ICP6 and ICP47 were consistent with the theoretical results. The number of virus vector particles was 4.5 × 1010 VP.mL-1, and the infection activity was 4.3 × 107 pfu.mL-1. The Vero cells were infected with HSV1 / GM-CSF for 72 h at a MOI of 0.01. The expression of GM-CSF in the supernatant was 228 ng.mL-1 after 72 h. The biological activity of the expressed product was 4.3 × 103 U.mL-1. The MOI IC50 of this gene therapy preparation against human breast cancer cell MCF7 in vitro was 0.08. The proliferation ratio of HSV1 / GM-CSF in human breast cancer cell line MCF7 and human embryo lung fibroblast diploid cell MRC5 was 308 at the same MOI and by pfu assay. No wild-type herpes simplex virus was detected. Other indicators are in line with “human gene therapy research and preparation of quality control technology guidelines” and the 2005 edition of “Chinese Pharmacopoeia” (three) requirements. Conclusion The quality control methods and quality standards of HSV1 / GM-CSF were initially established and used for the quality control of this product.