摘要
目的:构建全长人β-珠蛋白基因慢病毒载体,稳定转染K562细胞使得β-珠蛋白高效表达,为β-珠蛋白基因添加治疗奠定基础。方法:采用PCR扩增人正常β-珠蛋白基因,加上Sph I和Not I两个酶切位点进行T载体克隆,获得重组质粒p UC57-β-globin。用限制性内切酶连接目的基因片段和慢病毒载体,筛选获得慢病毒表达载体LV-β-globin,测序。将慢病毒载体转染293T细胞,包装病毒。用LV-β-globin感染K562细胞。采用流式细胞仪检测荧光效率,运用实时PCR及Western Blotting检测K562细胞中β-珠蛋白mRNA及蛋白表达情况。结果:通过PCR获得长度为2 128 bp的目的基因。连接p UC57载体和慢病毒表达载体,慢病毒表达载体LV-β-globin构建成功,序列正确。LV-β-globin转染K562细胞72 h后,显示大量绿色荧光表达,荧光效率为94.8%。β-珠蛋白mRNA 2-ΔΔCt值为4.080±0.078,高于对照组;β-珠蛋白灰度值为1.063±0.082,高于对照组,差异均有统计学意义(P<0.05)。结论:成功构建并包装含人β-珠蛋白基因的慢病毒载体并高效稳定表达。
Objective： To construct human β- globin gene lentiviral vector,transfect it into K562 cells to ensure high- efficiency expression of β- globin,lay the foundation for β- globin gene addition therapy. Methods： PCR was used to amplify human normal β- globin gene,T vector cloning was performed in Sph I and Not I restriction enzyme cutting sites to obtain recombinant plasmid p UC57- β- globin. Target gene segments and lentivirus vector were joined by restriction endonuclease,LV- β- globin was obtained after screening,then gene sequencing was conducted. Lentiviral vector was transfected into 293 T cells,then lentivirus was packaged. LV- β- globin was used to infect K562 cells. Flow cytometry was used to detect fluorescence efficiency,real- time PCR and Western Blotting were used to detect the expressions of β- globin mRNA and protein in K562 cells. Results： A length of 2 128 bp target gene sequence was obtained by PCR.p UC57 vector was connected to lentiviral expression vector. LV- β- globin was constructed successfully,the gene sequence was correct.LV- β- globin was transfected into K562 cells,after 72 hours,large green fluorescence was observed,fluorescence efficiency was 94. 8%.β- globin mRNA 2^-ΔΔCt value was 4. 080 ± 0. 078,which was higher than that in control group; grey value of β- globin was 1. 063 ± 0. 082,which was higher than that in control group,there were statistically significant differences（ P〈0. 05）. Conclusion： Lentiviral vector containing human β- globin gene is constructed and packaged successfully,and its expression is efficient and stable.
出处
《中国妇幼保健》
CAS
2015年第3期427-431,共5页
Maternal and Child Health Care of China
基金
广西科技基础条件平台建设项目〔11-031-07
12-071-05
13-051-12〕
广西医疗卫生重点科研课题〔2012061〕
广西医学科学实验中心开放基金专项项目〔KFJJ2010-15〕
