重组腺相关病毒rAAV-NT4-ADNF-9的构建及其对体外培养的大鼠耳蜗组织的转染

Construction of a recombinate adeno-associated virus rAAV-NT4-ADNF-9 and its transfection to the cultured cochleae of rats

目的:包装携载神经营养素(NT4)与活性依赖性神经营养因子-9(ADNF-9)融合基因的重组腺相关病毒rAAV-NT4-ADNF-9,并观察该重组病毒对体外培养的耳蜗组织的转染。方法:使用pSSHG-CMV-NT4-ADNF-9,pGF140和pAAV/Ad 3种质粒共转染293包装细胞,制备ADNF-9重组腺相关病毒(recomb inate adeno-assoc iated virus,rAAV),应用斑点杂交实验检测重组病毒滴度;体外分离培养新生SD大鼠的耳蜗毛细胞;将重组病毒感染体外培养的新生SD大鼠耳蜗毛细胞,于24 h后提取组织行反转录-聚合酶链式反应(RT-PCR)以检测rAAV-NT4-ADNF-9对耳蜗的转染。结果:应用斑点杂交实验检测重组病毒滴度为2×1013cfu/m l,表明成功地构建了重组AAV载体。成功分离培养新生SD大鼠的耳蜗毛细胞后,经RT-PCR反应,琼脂糖凝胶电泳检测结果显示NT4-ADNF-9在耳蜗组织得到了表达。结论:构建的重组病毒rAAV-NT4-ADNF-9,在体外可成功转染耳蜗组织,用于基因治疗的研究。

Objective： To construct an universal recombinate adeno-associated virus （AAV）, rAAV- NT4-ADNF-9, and to detect the ability of transfection of the rAAV vector into cochlea in vitro. Methods： pSSVHG-CMV-ADNF-9 plasmid was introduced into 293 cell by method of Ca3 （ PO4 ） 2 using three plasmids of pSSHG-CMV-NT4-ADNF-9, pGF140 and pAAV/Ad. The recombinate adeno-associated virus （rAAV） was harvested, and the titrations of the rAAV concentrated was detected by dot-blot test. To isolate and culture the cochlear hair cell of SD rats newly born in vitro. The rAAV-NT4-ADNF-9 was added to the medium while plating. Cochlears were collected 24 h after cultivation for RT-PCR to detect the transfection of rAAV- NT4-ADNF-9. Results： The titration of rAAV stock produced 2 × 10^13 total particles/ml, which showed that rAAV-NT4-ADNF-9 was constructed successfully. The cochlear hair cell of SD rats newly born was isolated and cultured in vitro successfully. It certified that rAAV-NT4-ADNF-9 was able to transfect into cochlear and express secretory NT4-ADNF-9 peptide by RT-PCR. Conclusion： The rAAV vector constructed in this paper, rAAV-NT4-ADNF-9, can transfer into cochlear cultured in vitro, which ]ayed a foundation of further research for gene therapy.