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从人外周血中分离淋巴细胞,经PMA和钙离子载体A23187诱导,通过RT-PCR技术获得hIL-5cDNA片段。扩增其编码成熟多肽的cDNA片段,插入pBV220,在大肠杆菌DH5α中诱导表达,未能得到预期14kD的重组蛋白的表达,只有一个克隆高效表达约10kD的小肽。对该克隆测序表明,其cDNA缺失一个A,导致在86位氨基酸处开始移码,高效表达一个93个氨基酸的小肽。这表明86位氨基酸之后的两处连续稀有密码子可能抑制了hIL-5cDNA在大肠杆菌的表达。通过重组PCR技术改变86位氨基酸之后稀有密码子的偏好性,在大肠杆菌中获得hIL-5重组蛋白的高效表达,表达量约占菌体总蛋白的15%,经免疫学鉴定为hIL-5重组蛋白
Lymphocytes were isolated from human peripheral blood, induced by PMA and calcium ionophore A23187, and hIL-5 cDNA fragments were obtained by RT-PCR. The cDNA fragment encoding the mature polypeptide was amplified, inserted into pBV220, and induced in E. coli DH5α. The expected 14kD recombinant protein expression was not obtained. Only one clone could efficiently express small peptide of about 10kD. Sequencing of this clone revealed that its cDNA lacks an A, resulting in a frame shift beginning at amino acid 86 and efficient expression of a 93-amino-acid small peptide. This suggests that two consecutive rare codons following amino acid 86 may inhibit the expression of hIL-5 cDNA in E. coli. The recombinant protein hIL-5 was expressed in Escherichia coli with a high level of expression of hIL-5 protein, accounting for about 15% of the total bacterial proteins. The recombinant protein was identified as hIL-5 Recombinant protein