中文摘要：

目的构建β-神经生长因子（β-NGF）慢病毒表达载体p LVX-TRE3G-IRES-β-NGF,应用Tet-on 3G四环素诱导表达系统,探讨β-NGF在人胚肾（HEK293FT）细胞中的过表达情况,及其对大鼠肾上腺嗜铬细胞瘤（PC12）细胞分化的影响。方法从SD大鼠脑组织提取总RNA,反转录成c DNA,利用分子生物学技术,克隆鼠β-NGF基因完整编码区,将β-NGF基因定向插入载体p LVX-TRE3G-IRES中。将重组载体p LVX-TRE3G-IRES-β-NGF和载体p LVX-Tet3G分别转染空白HEK293FT细胞,制备收获慢病毒,共转染HEK293FT细胞,同时用不同剂量强力霉素（Dox）诱导NGF表达（分为未转染组、0、100、500和1000μg/L Dox诱导表达组）。48h后收集细胞,免疫印迹法（Western blotting）检测各组细胞内β-NGF表达情况。同时收集培养上清,1.ELISA检测各组上清内β-NGF的分泌量;2.制作条件培养基,加入PC12细胞培养基中进行诱导培养,观察PC12细胞诱导分化情况。结果双酶切和测序鉴定重组质粒p LVX-TRE3G-IRES-β-NGF序列和方向正确;β-NGF在转染细胞内被诱导表达,随着Dox剂量增加,表达量增强;β-NGF在培养基的上清内表达,表达量随Dox剂量增加而增多。诱导表达的条件培养基可诱导PC12细胞形态改变,表达神经元特异性烯醇化酶（NSE）蛋白。结论成功重组慢病毒载体p LVX-TRE3G-IRES-β-NGF,转染后β-NGF基因能够在HEK293FT细胞中表达和分泌,且该蛋白具有诱导PC12细胞向神经元样细胞分化的能力;运用Tet-On 3G四环素诱导表达系统,可成功诱导表达获得不同剂量β-NGF蛋白。

英文摘要：

Objective To construct recombinant lentiviral vector carrying rat β-neural growth factor（ β-NGF）gene by inducing expression of the Tet-on 3G tetracycline,and to observe its expression in HEK293 FT cells and effects on PC12 cells. Methods Total RNA was extracted from rat brain as the template. By gene recombination technique,rat β-NGF gene was inserted into lentiviral vector as p LVX-TRE3G-IRES-β-NGF. The supernatant of virus including the recombinant vector of p LVX-TRE3G-IRES-β-NGF and p LVX-Tet3 G was collected from packaging cells,respectively,and then cotransfected blank HEK293 FT cells. The expression of β-NGF was induced with different doses of doxycycline（ Dox）（ divided into non-transfection group,0,100,500 and 1000μg/L of Dox inducing group）. Cells were collected and analyzed by Western blotting assay,48 hours after. The supernatant was detected by ELISA assay for the secretion and used as conditional medium for PC12 cells. Results A band of β-NGF gene was about 726 bp. After transfection,β-NGF protein was detected to express on 293 FT cells and supernatant following the increase of Dox dose. The neuron-specific enolase（ NSE） was expressed on PC12 cells after induced by conditional media. Conclusion That theβ-NGF gene was be expressed successfully on HEK 293 FT cells by Tet-on 3G induced expression system,and the protein has effected oninducing PC12 cells differentiation into neuron-like cells.