人白细胞介素

王　燕　朱锡华　万　瑛　纪贤文　刘　昕

　　中国图书分类号　R392.11
　　摘　要　目的：为了在大肠杆菌中高效表达hIL-3cDNA，获得hIL-3蛋白，拟对天然型hIL-3进行构建以获得新的突变体，同时进行了活性测定，以便深入了解hIL-3的结构与功能之间的关系。方法：利用PCR技术对天然型hIL-3 N末端第3位蛋氨酸(Met3)和C末端第17位蛋氨酸(Lys116)进行定点突变，获得突变体MhIL-3和MVhIL-3。结果：经Suger双脱氧法测序证实突变体 MhIL-3 cDNA,MVhIL-3 cDNA 的活性为6.9×104 U/ml，而天然型的是1.5×104 U/ml。天然型pSM53hNIL-3表达量占菌体总蛋白的7%，突变型pSM53MVhIL-3表达量占菌体总蛋白的16%。结论：人IL-3 cDNA可通过PCR技术进行定点突变，所获得的突变型pSM53 MVhIL-3活性比天然型pSM53hIL-3高3～4倍。
　　关键词　PCR定点突变　hIL-3 cDNA突变体　高水平表达　hIL-3突变体

PCR site-directed mutagenesis of human IL-3 cDNA in vitro human IL-3 by PCR site-directed mutagenesis study

WANG Yan, ZHU Xi-Hua①,WAN Ying et al.
Third Military Medical College,Molecular Biology Department, Chongqing 630038.①Third Military Medical College,Molecular Immunology Department, Chongqing 630038

　　　Abstract　Objective: In order to obtain a high level express of human IL-3,we modified the human IL-3 cDNA with PCR based mutagenesis method. And also we have determined the new mutant variant human IL-3 cDNA structure and its protein products activity. This method can be used to study gene modification, the structure or function of protein and the relationship between themselves.Methods: The human IL-3 cDNA mutants were obtained by PCR in vitro. The codon Met3 and Lys116 in human IL-3 cDNA were substituted by codon (GTT) of Val. Nucleotide sequences of the PCR products were determined by DNA sequence.Natural human IL-3 cDNA (NhIL-3 cDNA) and mutant variant human IL-3 cDNA (MvhIL-3 cDNA) have been inserted downstream of promoter with a set of expression vectors, and transfered into E.Coli.Results: E.Coli bacterial cells boring the plasmid pSM53 NhIL-3 expressed only 7% protein of the total bacterial body protein but the pSM53 MhIL-3 cDNA had produced a high level of human IL-3 about 16% of the total bacterial body protein. The molecular weight of the product is 15 kD. Their biological activity respectively is 1.5×

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