Gene cloning,prokaryotic vector constructing for the major allergen ovomucoid in Hen's egg white and its recombinant expression / 中国免疫学杂志

ABSTRACT

Objective:To clone and express the gene of ovomucoid,which is the main allevgen in egg white.Methods:Using total RNA of chicken oviduct as template,the gene of ovomucoid was amplified by RT-PCR.The homology was analyzed by comparision with the sequence in GeneBank.Subsequently,the PCR product of the ovomucoid gene was cloned into prokaryotic expressing vector pET-28a and was expressed by the challenge of IPTG.Results:The whole gene of ovomucoid,one of the main allergens in egg white,was successfully cloned.The cloned ORF sequence contains 633 bp,including stop codon,encods for 210 amino acids.Sequence analysis shows that the ovomucoid gene displays 99% nucleotide identities with the published sequences.The molecule weight of ovomucoid protein obtained was 21 kD.By the challenge of IPTG,SDS-PAGE analysis showed that the ovomucoid gene was overexpressed in E.coli BL21(DE3).Conclusion:The gene of ovomucoid is cloned and overexpressed in E.coli BL21(DE3).This study will be the basis for the further research.