人白细胞介素-3突变体的构建及在大肠杆菌中表达的研究

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

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３Ｎ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３ｃＤＮＡ，ＭＶｈＩＬ３ｃＤＮＡ的活性为６．９×１０４Ｕ／ｍｌ，而天然型的是１．５×１０４Ｕ／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３ｃＤＮＡ可通过ＰＣＲ技术进行定点突变。

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 1.5×10 4 U/ml and 6.9×10 4 U/ml。Conclusion:The human IL 3 cDNA can be reconstucted with PCR mutagenesis method to get a new human IL 3 cDNA mutant. The activity of this MVhIL 3 expressed protein we have obtained is 3 4 times of the NhIL 3.