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.
引文
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