title: Molecular Cloning of Human Interleukin-5 cDNA and Its Expression in E. coli.
abstract: hIL-5 cDNA was amplified through reverse transcription-polymerase chain reaction from peripheral blood lymphocytes induced with PMA and calcium ionophore A23187. The hIL-5 fragment was cloned into pUC18 and its sequence was identified to be hIL-5 cDNA sequence. The fragment which encodes hIL-5 mature polypeptide was amplified and cloned into pBV220 to express hIL-5 recombinant protein in E. coli, but there was no expected recombinant protein expressed. Only one clone expressed a 10kD peptide at high level. DNA sequencing showed that this clone had an adenosine deletion at 86th codon in hIL-5 cDNA and expressed a polypeptide of 93 amino acids at high level, and hIL-5 cDNA had not yet been expressed in E.coli successfully. These results suggested that two consecutive rare codons after 86th codon in hIL-5 cDNA could block polypeptide synthesis in E. coli. Through recombinant PCR technology the rare codons after 86th codon in hIL-5 cDNA were mutated into corresponding codons preferentially used in E. coli, and the mutated hIL-5 cDNA was highly expressed in pBVhIL5-H/DH5alpha to approximately 15% TCP after thermal induction. hIL-5 recombinant protein existed as inclusion bodies in E. coli. ELISA with a cross-reacting rat anti-mIL-5 monoclonal antibody confirmed the expressed product was hIL-5 recombinant protein.
authors.affiliation: Department of Immunology, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100850, China. gaojy@nic.bmi.ac.cn
authors.forename: Jun-Jie
authors.initials: JJ
authors.lastname: Mei
authors.forename: Jie-Ying
authors.initials: JY
authors.lastname: Gao
authors.forename: Guang-Zhi
authors.initials: GZ
authors.lastname: Ding
authors.forename: Yu-Hong
authors.initials: YH
authors.lastname: Liu
authors.forename: Hong
authors.initials: H
authors.lastname: Peng
authors.forename: Xiang-Ying
authors.initials: XY
authors.lastname: Kong