[1]周海燕,薛雨顺,王新川,等.人晶状体上皮细胞BHMT基因过表达慢病毒载体的构建及鉴定*[J].陕西医学杂志,2019,(8):963-967.
　ZHOU Haiyan,XUE Yushun,WANG Xinchuan,et al.Construction and identification of lentiviral vector for over-expressing BHMTgene and transfection into human lens epithelial cells in vivo[J].,2019,(8):963-967.
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人晶状体上皮细胞BHMT基因过表达慢病毒载体的构建及鉴定*
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《陕西医学杂志》[ISSN:1000-7377/CN:61-1281/TN]

卷:
期数:
2019年8期
页码:
963-967
栏目:
基础研究
出版日期:
2019-08-05

文章信息/Info

Title:
Construction and identification of lentiviral vector for over-expressing BHMTgene and transfection into human lens epithelial cells in vivo
文章编号:
DOI:10.3969/j.issn.1000-7377.2019.08.001
作者:
周海燕1; 薛雨顺1; 王新川2; 高宁宁
1.陕西省人民医院眼科（西安 710068）;2.空军军医大学基础医学院（西安 710032）
Author(s):
ZHOU Haiyan; XUE Yushun; WANG Xinchuan;  et al.
Department of Ophtalmology，Shaanxi Provincial People’s Hospital（Xi’an 710068）
关键词:
慢病毒载体; 人晶状体上皮细胞; 甜菜碱高半胱氨酸甲基转移酶; 过表达; 构建;  鉴定
Keywords:
Key words Lentivirus;  Human lens epithelial cells; Betaine-homocysteinemethy transferase; Over- expression;  Construction;  Identification
分类号:
R365
文献标志码:
A
摘要:
摘 要 目的：构建甜菜碱高半胱氨酸甲基转移酶（BHMT）基因过表达慢病毒载体，并检测其在人晶状体上皮细胞中的表达效果。方法：采用聚合酶链反应（PCR）扩增制备目的基因片段，构建BHMT慢病毒过表达载体,转染至293T细胞。运用免疫荧光法行病毒滴度检测，并利用荧光显微镜观察细胞中绿色荧光蛋白的表达,运用RT-PCR、Western blot检测鉴定BHMT mRNA 蛋白的表达水平。将构建成功的慢病毒载体BHMT-GV358感染人晶状体上皮细胞,观察目的基因表达。结果：重组慢病毒转染 293T 细胞后可观察到荧光,在稳定转染的细胞中 BHMT及蛋白的表达水平较未转染细胞显著增高(P＜0.05),体外转染人晶状体上皮细胞，荧光显微镜下可见 BHMT 表达。结论：成功构建过表达BHMT的慢病毒载体,并有效感染人晶状体上皮细胞,为进一步研究 BHMT在白内障发生发展中的作用提供生物学基础。
Abstract:
Abstract Objective: To obtain lentiviral vector for over- expressing BHMT gene and transfected human lens epithelial cells in vivo.Methods: The whole length BHMT gene fragment was obtained a by polymerase chain reaction(PCR).The lentiviral overexpression vector of BHMT was constructed ,then the recombinants were transfected into 293T cells with Lipofect 2000 Reagent. The titer was determined by fluorescence method. BHMT was monitored by using fluorescence microscope and the mRNA and protein expression of BHMT was detected by RT- PCR and western blot. The human lens epithelial cells were transfected by the lentivirus vectors of BHMT- GV358 in vivo.Results：A large number of significant fluorescent cells could be observed after recombinant lentiviruses were co-transfected into 293T cells. RT-PCR showed that the expression level of BHMT was significantly increased (P＜0.05). The expression of BHMT protein could be seen with the inverted fluorescence microscope after the human lens epithelial cells were infected by the lentivirus vectors.Conclusion: The lentiviral vector of over- expression BHMT gene is constructed successfully, and can transfected human lens epithelial cells effectively in vivo. which provides an experimental foundation for further studies about the relationship between BHMT and cataract.

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备注/Memo

备注/Memo:
*国家自然科学基金资助项目(81600720） 陕西省自然科学基金资助项目（2017JQ8012）
更新日期/Last Update: 2019-08-13