寡聚脱氧核苷酸介导的体外定点突变校正VEGFcDNA

目的：对由ＰＣＲ扩增的血管内皮细胞生长因子（ＶＥＧＦ１６５）ｃＤＮＡ编码Ｎ－２４位氨基酸错误配对的密码子ＡＣＧ（应为ＡＧＣ丝氨酸）进行校正。方法：用一个化学合成的寡聚脱氧核苷酸片断作为诱变引物对ＶＥＧＦ１６５）ｃＤＮＡ编码Ｎ－２４位氨基酸错误配对的密码子进行体外定点突变。转化子经打点杂交及温度梯度洗膜，筛选出突变子，再ＤＮＡ序列分析。结果：ＶＥＧＦ１６５ｃＤＮＡ编码Ｎ－２４位氨基酸错误配对的密码子变为ＡＧＣ（丝氨酸）。结论：该突变方法可靠，省时，实用。

OLIGONUCLEOTIDE-DIRECTED SITE-SPECIFIC MUTAGENESIS FOR RECTIFING cDNA OF VASCULAR ENDOTHELIAL GROWTH FACTOR(VEGF)

To rectify utagenesis the vascular endothelial growth factor(VEGF165)cDNA which was ampli-fied from 18W human fetal brain cDNA library and showed a wrong codon of N-24 amono acid ACG/Thr wgich should beAGC/Ser. Method: With a mutagenetic primer and through sitespecific mutagenesis, the VEGF165 cDNA was rectified. Theprocedures included: the annealing of a single-stranded uracil-containing DNA template with oligonucleotide primer in vitro synthesis and ligation, transforming JM109 of E. Coli screening mutants by washing the filter at an increasing temper-ature and seqnencing. Result It was confirmed by the positive mutants the positive mutants was confirmed that codon N-24amino acid was AG C/Serine by DNA sequencing. Conclusion: This method is reliabe and pracicable