β-神经生长因子基因克隆和诱导表达及其对PC12细胞的诱导分化作用
发布时间：2019-06-05 20:04
【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。
[Abstract]:Objective to construct 尾-nerve growth factor (尾-NGF) lentivirus expression vector p LVX-TRE3G-IRES- 尾-NGF, and to investigate the overexpression of 尾-NGF in human embryonic kidney (HEK293FT) cells by using Tet-on 3G tetracycline induced expression system. And its effect on the differentiation of rat adrenal pheochromocytoma (PC12) cells. Methods Total RNA, was extracted from the brain tissue of SD rats and transcribed into c DNA,. The complete coding region of mouse 尾-NGF gene was cloned by molecular biology technique, and the 尾-NGF gene was inserted into vector p LVX-TRE3G-IRES. The recombinant vector p LVX-TRE3G-IRES- 尾-NGF and vector p LVX-Tet3G were transfected into blank HEK293FT cells respectively. Lentivirus was prepared and co-transfected into HEK293FT cells. NGF expression was induced by different doses of doxycycline (Dox) (divided into untransfected group). 0100500 渭 g / L and 1000 渭 g / L Dox induced expression groups). 48 hours later, the cells were collected and the expression of 尾-NGF in each group was detected by immunoblotting (Western blotting). At the same time, the culture supernatant was collected and the secretion of 尾-NGF in each group was detected by 1.ELISA. The conditioned medium was prepared and cultured in PC12 cell medium to observe the differentiation of PC12 cells. Results the sequence and direction of the recombinant plasmid p LVX-TRE3G-IRES- 尾-NGF were identified by double enzyme digestion and sequencing, and the expression of 尾-NGF was induced in the transfected cells, and the expression increased with the increase of Dox dose. 尾-NGF was expressed in the culture medium, and the expression increased with the increase of Dox dose. The conditioned medium could induce the morphological changes of PC12 cells and express neuron-specific enolase (NSE) protein. Conclusion the recombinant lentivirus vector p LVX-TRE3G-IRES- 尾-NGF, can express and secrete 尾-NGF gene in HEK293FT cells, and the protein can induce PC12 cells to differentiate into neuron-like cells. Different doses of 尾-NGF protein could be successfully induced by Tet-On 3G tetracycline induced expression system.
【作者单位】： 贵州医科大学组织学胚胎学教研室;贵州医科大学贵州省细胞工程生物医药技术国家地方联合工程实验室贵州省再生医学重点实验室;
【基金】：贵州省优秀科技教育人才省长专项资金[黔省专合字(2012)41号] 贵州省科技厅-贵阳医学院联合基金[黔科合LG字(2012)026号]
【分类号】：R772.2
[Abstract]:Objective to construct 尾-nerve growth factor (尾-NGF) lentivirus expression vector p LVX-TRE3G-IRES- 尾-NGF, and to investigate the overexpression of 尾-NGF in human embryonic kidney (HEK293FT) cells by using Tet-on 3G tetracycline induced expression system. And its effect on the differentiation of rat adrenal pheochromocytoma (PC12) cells. Methods Total RNA, was extracted from the brain tissue of SD rats and transcribed into c DNA,. The complete coding region of mouse 尾-NGF gene was cloned by molecular biology technique, and the 尾-NGF gene was inserted into vector p LVX-TRE3G-IRES. The recombinant vector p LVX-TRE3G-IRES- 尾-NGF and vector p LVX-Tet3G were transfected into blank HEK293FT cells respectively. Lentivirus was prepared and co-transfected into HEK293FT cells. NGF expression was induced by different doses of doxycycline (Dox) (divided into untransfected group). 0100500 渭 g / L and 1000 渭 g / L Dox induced expression groups). 48 hours later, the cells were collected and the expression of 尾-NGF in each group was detected by immunoblotting (Western blotting). At the same time, the culture supernatant was collected and the secretion of 尾-NGF in each group was detected by 1.ELISA. The conditioned medium was prepared and cultured in PC12 cell medium to observe the differentiation of PC12 cells. Results the sequence and direction of the recombinant plasmid p LVX-TRE3G-IRES- 尾-NGF were identified by double enzyme digestion and sequencing, and the expression of 尾-NGF was induced in the transfected cells, and the expression increased with the increase of Dox dose. 尾-NGF was expressed in the culture medium, and the expression increased with the increase of Dox dose. The conditioned medium could induce the morphological changes of PC12 cells and express neuron-specific enolase (NSE) protein. Conclusion the recombinant lentivirus vector p LVX-TRE3G-IRES- 尾-NGF, can express and secrete 尾-NGF gene in HEK293FT cells, and the protein can induce PC12 cells to differentiate into neuron-like cells. Different doses of 尾-NGF protein could be successfully induced by Tet-On 3G tetracycline induced expression system.
【作者单位】： 贵州医科大学组织学胚胎学教研室;贵州医科大学贵州省细胞工程生物医药技术国家地方联合工程实验室贵州省再生医学重点实验室;
【基金】：贵州省优秀科技教育人才省长专项资金[黔省专合字(2012)41号] 贵州省科技厅-贵阳医学院联合基金[黔科合LG字(2012)026号]
【分类号】：R772.2
【相似文献】
相关期刊论文 前8条
1 郑雅娟;辛华;张文松;王丹;;氯离子通道阻滞剂对人RPE细胞增生抑制作用的实验研究[J];眼科研究;2006年04期
2 张文一,白海青,王z牖，
本文编号：2493791
本文链接：https://www.wllwen.com/yixuelunwen/wuguanyixuelunwen/2493791.html
最近更新
教材专著
