Production of Equine Infectious Anemia Virus (EIAV) antigen in Pichia pastoris

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• 作者：Luciana Cavalcanti de Arruda Coutinho^a ; ^{lcacoutinho@gmail.com} ; André ; Luiz Santos de Jesus^b ; ^{andreluizsjesus@gmail.com} ; Karin Florê ; ncio Lins de Paiva Fontes^a ; ^{karinffontes@gmail.com} ; Eliane Campos Coimbra^b ; ^{elianeccoimbra@gmail.com} ; Filipe Colaç ; o Mariz^b ; ^{filipe.mariz@yahoo.com.br} ; Antonio Carlos de Freitas^b ; ^{acf_ufpe@yahoo.com.br} ; Rita de Cá ; ssia Carvalho Maia^a ; ^{rccmaia@dmv.ufrpe.br} ; Roberto Soares de Castro^a ; ^{rscastro@dmv.ufrpe.br}
• 关键词：Methylotrophic yeasts ; Western blotting ; AGID ; Capsid protein ; ELISA ; EIA
• 刊名：Journal of Virological Methods
• 出版年：2013
• 出版时间：August, 2013
• 年：2013
• 卷：191
• 期：2
• 页码：95-100
• 全文大小：914 K

文摘

Equine Infectious Anemia (EIA) is a persistent lentivirus infection of horses which causes a chronic clinical condition with worldwide importance in veterinary medicine. The p26 protein is usually prepared for use as an antigen in serological tests for EIA diagnosis since it is a well-conserved gene sequence and very immunogenic. In view of the ability of yeast to make post-translational modifications of proteins, this study was carried out to allow Pichia pastoris to be used for the expression of a synthetic codon-optimized EIAV p26 gene. The gene was cloned into pPICZ¦ÁA vector after appropriate enzymatic digestion. P. pastoris clones transformed with the pPICZ¦ÁAp26 construction were induced to produce the recombinant p26 protein (rp26) under the regulation of alcohol oxidase 1 promoter by adding methanol to the culture medium. The p26 gene expression was detected by RT-PCR and the production of rp26 was confirmed by dot blotting, Western blotting, ELISA and AGID. The P. pastoris expression system was capable of producing a functional EIAV p26 protein that can be used directly in the functionality tests without requiring laborious purification or recovery steps. This is the first reported study of EIAV p26 protein production in yeast cells.