CAJ | 학술논문

目的建立高效的临床级重组腺相关病毒( rAAV)制备方法.方法将rAAV的反向末端重复序列( ITR)及目的基因表达框(2053 bp)从pAAV-MCS剪切引入杆状病毒载体中构建顺式载体,rAAV血清型2的衣壳和复制蛋白基因在反式载体pFastBacDual中利用真核基因遗漏扫描原理表达,两者分别制备成杆状病毒,共感染昆虫细胞sf9包装rAAV.增强绿色荧光蛋白(EGFP)作为报告基因验证其可行性.结果顺式和反式杆状病毒载体分别在DH 10BacTM中转座形成杆粒bacmid、昆虫细胞sf9中制备成杆状病毒,病毒滴度可达1×108～3 ×108 v.g./ml.二杆状病毒共感染昆虫细胞sf9,完成rAAV拯救、复制和包装过程,经rAAV-EGFP感染293T细胞测试具有良好的生物学活性.结论该系统具有高效、安全、简便等特点,为临床级rAAV制备和基因治疗奠定了基础.
목적 건립고효적림상급중조선상관병독( rAAV)제비방법.방법 장rAAV적반향말단중복서렬( ITR)급목적기인표체광(2053 bp)종pAAV-MCS전절인입간상병독재체중구건순식재체,rAAV혈청형2적의각화복제단백기인재반식재체pFastBacDual중이용진핵기인유루소묘원리표체,량자분별제비성간상병독,공감염곤충세포sf9포장rAAV.증강록색형광단백(EGFP)작위보고기인험증기가행성.결과 순식화반식간상병독재체분별재DH 10BacTM중전좌형성간립bacmid、곤충세포sf9중제비성간상병독,병독적도가체1×108～3 ×108 v.g./ml.이간상병독공감염곤충세포sf9,완성rAAV증구、복제화포장과정,경rAAV-EGFP감염293T세포측시구유량호적생물학활성.결론 해계통구유고효、안전、간편등특점,위림상급rAAV제비화기인치료전정료기출.
Objective To establish an efficient and safe method for production of recombinant adeno-associated viruses (rAAV) of clinical scale.Methods The 2053 bp of rAAV inverted terminal repeat (ITR) and foreign gene expression cassette was cloned into baculovirus vector as cis-acting vector.The capsid and replication proteins of rAAV serum type 2 were expressed in a trans-acting baculovirus vector (pFastBacDual) by leaky scanning mechanism of eukaryotic gene transcription.Both constructs were made into baculovirus which infected insect cells st9 for packaging rAAV.Enhanced green fluorescent protein (EGFP) was used as reporter gene to verify the feasibility of system.Results Transposition of cis-acting and trans-acting vector into bacmid was carried out in DH10BacTM reswctively.Two baculoviruses were prepared in insect cells sf9 and the titers were about 1×108-3×108 v.g./mL.rAAV were rescued,replicated and packaged by co-infection of two baculoviruses into sf9.The biological activity of rAAV-EGFP was proved by infecting 293T cells.Conclusion This system provides an experimental data for making rAAV of clinical scale and a good basis for future gene therapy.