UGT1A1基因Q239X突变型过表达慢病毒载体的构建及病毒包装
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摘要
目的构建尿苷二磷酸葡萄糖醛酸转移酶1A1(UGT1A1)基因Q239X突变型过表达慢病毒载体并进行慢病毒包装。方法设计合成UGT1A1基因Q239X突变型片段,将纯化的UGT1A1基因Q239X突变型片段与线性化的慢病毒表达载体GV358 DNA进行连接反应,构建GV358-UGT1A1(p. Q239X)慢病毒表达载体。利用PCR及DNA测序鉴定GV358-UGT1A1(p. Q239X)阳性克隆;将构建成功的GV358-UGT1A1(p. Q239X)慢病毒表达载体和辅助包装质粒共转染293T细胞,进行病毒包装,并用梯度稀释法测定病毒滴度。结果通过PCR技术成功地扩增UGT1A1基因Q239X突变型片段并连接到GV358载体上,PCR鉴定及DNA测序结果示GV358-UGT1A1(p. Q239X)慢病毒表达载体构建成功。转染293T细胞后可观察到绿色荧光蛋白表达,测定其浓缩病毒滴度为2×108TU/m L。结论 UGT1A1基因Q239X突变型过表达慢病毒载体构建成功,包装获得高滴度慢病毒,为UGT1A1基因的后续研究奠定了基础。
Objective To construct a lentiviral vector over-expressing uridine diphosphate glucuronyltransferase 1 A1( UGT1 A1) gene Q239 X mutant and to package it into the viral particles. Methods The UGT1 A1 gene Q239 X mutant fragment was designed and synthesized. The purified UGT1 A1 gene Q239 X mutant fragment was ligated with the linearized lentiviral expression vector GV358 DNA to construct GV358-UGT1 A1( p. Q239 X) lentiviral expression vector. Positive clones of GV358-UGT1 A1( p. Q239 X) were identified by PCR and DNA sequencing; the 293 T cells were co-transfected with the successfully constructed GV358-UGT1 A1( p. Q239 X) lentiviral expression vector and the packaging plasmids.The virus titer was determined by virus gradient dilution method. Results The UGT1 A1 gene Q239 X mutant fragment was successfully amplified by PCR and ligated into the GV358 vector. The PCR and DNA sequencing results indicated that the GV358-UGT1 A1( p. Q239 X) lentiviral vector was successfully constructed. After transfecting 293 T cells,its green fluorescent protein expression was observed and the virus titer was 2 × 108 TU/m L. Conclusion The lentiviral vector over-expressing UGT1 A1 gene Q239 X mutant is successfully constructed with a high yield of lentivirus,and lays the foundation for further study of UGT1 A1 gene.
Objective To construct a lentiviral vector over-expressing uridine diphosphate glucuronyltransferase 1 A1( UGT1 A1) gene Q239 X mutant and to package it into the viral particles. Methods The UGT1 A1 gene Q239 X mutant fragment was designed and synthesized. The purified UGT1 A1 gene Q239 X mutant fragment was ligated with the linearized lentiviral expression vector GV358 DNA to construct GV358-UGT1 A1( p. Q239 X) lentiviral expression vector. Positive clones of GV358-UGT1 A1( p. Q239 X) were identified by PCR and DNA sequencing; the 293 T cells were co-transfected with the successfully constructed GV358-UGT1 A1( p. Q239 X) lentiviral expression vector and the packaging plasmids.The virus titer was determined by virus gradient dilution method. Results The UGT1 A1 gene Q239 X mutant fragment was successfully amplified by PCR and ligated into the GV358 vector. The PCR and DNA sequencing results indicated that the GV358-UGT1 A1( p. Q239 X) lentiviral vector was successfully constructed. After transfecting 293 T cells,its green fluorescent protein expression was observed and the virus titer was 2 × 108 TU/m L. Conclusion The lentiviral vector over-expressing UGT1 A1 gene Q239 X mutant is successfully constructed with a high yield of lentivirus,and lays the foundation for further study of UGT1 A1 gene.
引文
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[11]冯于玲,高宗燕,刘义,等.Crigler-NajjarⅠ型综合征患者及其家系UGT1A1基因的遗传分析[J].中华实用儿科临床杂志,2014,29(11):847-850.
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[14]孟凡荣,陈琛,万海粟,等.慢病毒载体及其研究进展[J].中国肺癌杂志,2014,17(12):870-876.
[15]辛凌翔,常迪.慢病毒载体表达系统的研究进展[J].中国动物保健,2017,19(10):84-86.
[2]Amandito R,Putradista R,Jikesya C,et al.UGT1A1 gene and neonatal hyperbilirubinemia:a preliminary study from Bengkulu,Indonesia[J].BMC Research Notes,2018,11(1):172.
[3]王小琴,华子瑜.UGT1A1基因多态性与新生儿高胆红素血症相关性研究现状[J].儿科药学杂志,2015,21(11):58-61.
[4]Canu G,Minucci A,Cecilia Z,et al.Gilbert and crigler najjar syndromes:an update of the UDP-glucuronosyltransferase 1A1(UGT1A1)gene mutation database[J].Blood Cells Mol Dis,2013,50(4):273-280.
[5]韦小兰,骆子义.新生儿不明原因高胆红素血症与UGT1A1基因多态性的研究进展[J].实用临床医学,2017,18(1):101-105.
[6]孙碧君,赵诸慧,杨琳,等.UGT1A1基因G71R突变与新生儿不明原因严重高胆红素血症相关[J].中国循证儿科杂志,2015,10(1):29-33.
[7]侯国强,王莉,阴怀清.新生儿重症高胆红素血症与UGTl A1基因多态性的相关性研究[J].中国新生儿科杂志,2016,31(4):247-250.
[8]Gong QH,Cho JW,Huang T,et al.Thirteen UDP glucuronosyltransferase genes are encoded at the human UGT1 gene complex locus[J].Pharmacogenetics,2001,11(4):357-368.
[9]Walter BK.Vertebrate UDP-glucuronosyltransferases:functional and evolutionary aspects[J].Biochem Pharmacol,2003,66(5):691-696.
[10]孙革,杜立中.尿苷二磷酸葡萄糖醛酸转移酶1A1基因与新生儿黄疸[J].中华儿科杂志,2006,44(1):71-73.
[11]冯于玲,高宗燕,刘义,等.Crigler-NajjarⅠ型综合征患者及其家系UGT1A1基因的遗传分析[J].中华实用儿科临床杂志,2014,29(11):847-850.
[12]Thrasher AJ.Progress in lentiviral vector technologies[J].Hum Gene Ther,2013,24(2):117-118.
[13]Cockrell AS,Kafri T.Gene delivery by lentivirus vectors[J].Mol Biotechnol,2007,36(3):184-204.
[14]孟凡荣,陈琛,万海粟,等.慢病毒载体及其研究进展[J].中国肺癌杂志,2014,17(12):870-876.
[15]辛凌翔,常迪.慢病毒载体表达系统的研究进展[J].中国动物保健,2017,19(10):84-86.