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目的建立搭载2型人成纤维细胞生长因子(human fibroblast growth factor 2,h FGF2)重组F基因缺失型仙台病毒(Sendai virus,Se V)制剂(Se V-h FGF2/d F)的质控方法与质量标准。方法采用RT-PCR法对Se V载体中插入的目的基因h FGF2和缺失的F基因进行鉴别,同时扩增两个基因片段M-HN和h FGF2-NP,以鉴别该病毒;鸡红细胞凝集试验检测制品的血凝效价,并计算病毒颗粒数;免疫荧光法测定Se V-h FGF2/d F的感染滴度,结合病毒颗粒数计算其比滴度;Se V-h FGF2/d F体外感染COS7细胞后,ELISA法测定感染上清中h FGF2的表达量;将感染上清体外作用于BALB/c 3T3细胞,3T3细胞增殖法检测h FGF2的生物学活性;酚-氯仿法抽提样品的DNA,Pico-Green法测定制品中DNA残留量;PCR法检测腺病毒残留。结果重组Se V-h FGF2/d F基因组中h FGF2基因、F基因、M-HN片段和h FGF2-NP片段的RT-PCR鉴定结果与理论值相符;血凝效价为1∶512,病毒颗粒数为4.12×1010 VP/ml;感染滴度为2.06×109 CIU/ml,比滴度为5.0%;Se V-h FGF2/d F以MOI 10感染COS7细胞72 h后,ELISA检测上清中h FGF2的表达量为117.1 ng/ml;表达上清中h FGF2的生物学活性为437 IU/ml;DNA残留量为2.24 ng/ml;制品中腺病毒的PCR检测结果为阴性;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《中国药典》三部(2010版)要求。结论初步建立了Se V-h FGF2/d F的质控方法和质量标准,为保证该制品的安全、有效、质量可控奠定了基础,同时也为其他Se V载体基因治疗药物的质量控制提供了参考。
OBJECTIVE: To establish a quality control method and quality control method for Se Vh FGF2 / d F strain carrying recombinant F gene of human fibroblast growth factor 2 (h FGF2) standard. Methods RT-PCR was used to identify the h FGF2 gene and the deleted F gene inserted into the Se V vector. Two gene fragments, M-HN and h FGF2-NP, were amplified to identify the virus. The chicken erythrocyte agglutination test The titer of Se Vh FGF2 / d F was measured by immunofluorescence and the specific titer was calculated with the number of virus particles. After in vitro infection of COS7 cells with Se Vh FGF2 / d F, The expression of hFGF2 in the supernatant was detected by ELISA. The supernatant of BALB / c 3T3 cells was infected with the supernatant of the infected cells. The biological activity of hFGF2 was detected by 3T3 cell proliferation assay. The DNA, Pico -Green method to determine the DNA residues in the product; PCR method to detect adenovirus residues. Results The RT-PCR results of h FGF2 gene, F gene, M-HN fragment and h FGF2-NP fragment in the recombinant Se Vh FGF2 / d F genome were consistent with the theoretical values. The titer of blood coagulation was 1:512 and the number of virus particles The infection titer was 2.06 × 109 CIU / ml and the specific titer was 5.0%. After COS7 cells were infected with Se Vh FGF2 / d F at MOI 10 for 72 h, the expression of hFGF2 The expression level was 117.1 ng / ml. The biological activity of hFGF2 in the supernatant was 437 IU / ml. The residual DNA was 2.24 ng / ml. The PCR results of the adenovirus in the product were negative. “Human gene therapy research and preparation of quality control technology guidelines” and “Chinese Pharmacopoeia” three (2010 version) requirements. Conclusion The quality control method and quality standard of Se Vh FGF2 / d F were preliminarily established, which laid the foundation for the safe, effective and quality control of Se Vh FGF2 / d F and provided a reference for the quality control of other Se V vector gene therapy drugs. .