人脑源性神经营养因子及神经营养素-3 重组腺病毒的构建及鉴定

Construction and identification of recombinant adenovirus vectors expressing brain derived neurotrophic factor and neurotrophin 3

目的：构建人脑源性神经营养因子（ｂｒａｉｎｄｅｒｉｖｅｄ ｎｅｕｒｏｔｒｏｐｈｉｃ ｆａｃｔｏｒ， Ｂ Ｄ Ｎ Ｆ）和神经营养素３（ｎｅｕｒｏｔｒｏｐｈｉｎ３， Ｎ Ｔ３）重组腺病毒，使其携带神经营养因子感染神经系统细胞，在局部释放有生物活性的营养因子。方法：将人全长 Ｂ Ｄ Ｎ Ｆ与 Ｎ Ｔ３ 基因分别插入ｐ Ｃ Ｄ Ｎ Ａ３ 载体，然后将表达序列 Ｃ Ｍ Ｖ Ｂ Ｄ Ｎ Ｆ Ｂ Ｇ Ｈｐ Ａ 和 Ｃ Ｍ Ｖ Ｎ Ｔ３ Ｂ Ｇ Ｈｐ Ａ 切下，插入 Ｅ１ 区缺失的腺病毒载体 ｐ Ｈ△ Ｅ１ｓｐ１ Ａ 中，与ｐ Ｊ Ｍ１７ 共转染２９３ 细胞，通过同源重组产生重组腺病毒。结果和结论：经过二次 Ｃｓ Ｃｌ超离心，获得了滴度分别为８×１０１１和１×１０１２ｐｆｕ／ｍ ｌ的 Ｂ Ｄ Ｎ Ｆ及 Ｎ Ｔ３ 重组腺病毒，并能够感染８ 日龄鸡胚背根神经节组织块，可促进神经突起的生长。这一结果显示， Ｂ Ｄ Ｎ Ｆ及 Ｎ Ｔ３ 的重组腺病毒在感染细胞内可表达有活性的 Ｂ Ｄ Ｎ Ｆ和 Ｎ Ｔ３ 蛋白，表明其可能应用于神经系统的基因治疗。

Objective: To construct Ad BDNF and Ad NT3, two recombinant adenoviral vectors containing the BDNF cDNA and NT3 cDNA expression cassettes for delivering brain derived neurotrophic factor(BDNF) and neurotrophin 3(NT3) to the nervous system, respectively. Methods: The full length BDNF and NT3 cDNA were cloned into pCDNA3 vector, then the expression cassettes CMV BDNF BGHpA and CMV NT3 BGHpA were cut and inserted into E1 substituted adenovirus vector pH△E1sp1A. After co transfection into 293 cells with adenovirus plasmid pJM17, the recombinant adenoviruses Ad BDNF, Ad NT3 were propagated in 293 cells via homologous recombination. Results and Conclusion: After CsCl ultra centrifugation for two times, the human BDNF and NT3 recombinant adenoviruses were obtained with titers of 8×10 11 , 1×10 12 pfu/ml respectively. The Ad BDNF, Ad NT3 could transfect and promote the neurite outgrowth of E8 dorsal root ganglion neuron. The result shows that the BDNF, NT3 recombinant adenovirus can express biologically active BDNF, NT3 and be potentially used in the gene therapy for nervous system.