实时荧光定量PCR法用于重组猿免疫缺损病毒-人色素上皮衍生因子注射液中复制型SIV的检测

目的：建立重组猿免疫缺损病毒-人色素上皮衍生因子(simian immunodeficiency virus-human pigment epithelium-derived factor，SIV-hPEDF)注射液中复制型SIV的实时定量PCR检测方法。方法：在提取重组SIV-hPEDF注射液供试品基因组RNA后，采用实时定量PCR的方法，检测供试品中的复制型SIV。结果：经过3次实验，所得TAT RNA标准品标准曲线均线性良好，线性方程为Y=-3.248lgX+38.12，相关系数为0.994；重组SIV-hPEDF注射液供试品中复制型SIV检测结果均为阴性。结论：所建立的复制型SIV实时荧光定量PCR检测方法重复性好、灵敏、准确，可用于重组SIV-hPEDF注射液中复制型SIV的检测。

Objective: To develop a real-time PCR method for the determination of reproducible SIV in recombinant simian immunodeficiency virus-human pigment epithelium-derived factor(SIV-hPEDF)injection. Method: After extraction of the genome RNA in recombinant SIV-hPEDF injection,real-time PCR was used to determine reproducible SIV in test samples. Result: The 3parallel experiments showed the TAT RNA standard curves had good linearity(r>0.994),and the equation was Y=-3.248lgX+38.12.The Results were all negative in the determination of reproducible SIV in recombinant SIV-hPEDF injection. Conclusion: The established method is repeatable,sensitive and accurate,and applicable in the determination of reproducible SIV in recombinant SIV-hPEDF injection.

[1] Tombran-Tink J,Barnstable CJ.PEDF:a multifaceted neurotrophic factor[J].Nat Rev Neurosci,2003,4(8):628
[2] Becerra SP.Focus on molecules:poigment epithelium－derived factor(PEDF)[J].Exp Eye Res,2006,82(5):739
[3] Phillips NJ,Ziegler MR,Radford DM,et al.Allelic deletion on chromosome 17p13.3 in early ovarian cancer[J].Cancer Res,1996,56(3):606
[4] Takita H,Yoneya S,Gehlbaeh PL,et al.Retinal neuroprotection against ischemic injury mediated by intraocular gene transfer of pigment epithelium-derived factor[J].Invest Ophthalmol Vis Sci,2003,44(10):4497
[5] Alcantara MB,Dass CR.Pigment epithelium-derived factor as a natural matrix metalloproteinase inhibitor:a comparison with classical matrix metalloproteinase inhibitors used for cancer treatment[J].J Pharm Pharmacol,2014,66(7):895
[6] ZHANG Zhong-hong(张忠红),LUAN Jie(栾洁).Intraocular neovascularization and cytokine(眼内新生血管与细胞因子)[J].IntJ Ophthalmol(国际眼科杂志),2006,6(1):158
[7] Bender B,Hoffmann OI,Negre D,et al.Low titer lentiviral transgenesis in rodents with simian immundeficiency virus vector[J].Biotechniques,2013,55(3):137
[8] Wunderlich S,Haase A,Merkert S,et al.Induction of pluripotent stem cells from a cynomolgus monkey using a polycistronic simian immunodeficiency virus-based vector,differentiation toward functional cardiomyocytes,and generation of stably expressing reporter lines[J].Cell Reprogram,2012,14(6):471
[9] Watanabe N,Ohashi K,Tatsumi K,et al.Genetically modified adipose tissue-derived stem/stromal cells,using simian immunodeficiency virus-based lentiviral vectors,in the treatment of hemophilia B[J].Hum Gene Ther,2013,24(3):283
[10] Daniel S.Novel viral vector systems for gene therapy[J].Viruses,2010,2(4):1002
[11] Hansen SG,Piatak MJ,Ventura AB,et al.Immune clearance of highly pathogenic SIV infection[J].Nature,2013,502(7469):100
[12] Tareen SU,Nicolai CJ,Campbell DJ,et al.A rev-independent gag/pol eliminates detectable psi-gag recombination in lentiviral vectors[J].Biores Open Access,2013,2(6):421
[13] Kuate S,Marino MP,Reiser J.Analysis of partial recombinants in lentiviral vector preparations[J].Hum Gene Ther Methods,2014,25(2):126
[14] LIU Yin(刘茵).Application of lentiviral vectors in transgenic animals development(慢病毒载体在转基因动物研制中的应用)[J].Int J Genet(国际遗传学杂志),2007,30(5):374
[15] WANG Lan(王兰),LI Yong-hong(李永红),RAO Chun-ming(饶春明).Determination of residual host genomic DNA in recombinant products by real-time PCR(实时定量PCR检测重组技术产品中宿主基因组DNA残留)[J].Chin J Pharm Anal(药物分析杂志),2009,29(10):1593
[16] MA Hai-yan(马海燕),FANG Yu-dan(方彧聃),ZHANG Jing-zhi(张敬之).A novel method for the determination of recombinant lentiviral titer and infectivity by qRT-PCR(应用荧光实时定量PCR方法检测重组慢病毒滴度及其感染效率)[J].Life Sci Res(生命科学研究),2009,13(5):394
[17] CHEN Yu(陈瑜),YANG Yao-yun(杨耀云),LU Shuai-yao(鲁帅尧),et al.Preparation and appraisal of the recombinant eGFP-containing lentiviral particles(携带eGFP基因慢病毒颗粒的制备及鉴定)[J].Biotechnology(生物技术),2013,23(6):76