Heterologous expression of Bluetongue VP2 viral protein fragment in Pichia pastoris

被引:11
作者
Athmaram, T. N.
Bali, Geetha [1 ]
Kahng, Goon G.
Dwarakanath, Sulatha
机构
[1] Bangalore Univ, Dept Microbiol & Biotechnol, Bangalore 560056, Karnataka, India
[2] Nano Sci Diagnost Inc, Austin, TX 78758 USA
[3] Jinju Natl Univ, Coll Agr & Biotechnol, Dept Food Sci, Chinju City 660758, South Korea
关键词
bluetongue virus serotype 23; Pichia pastoris; secreted BTV VP2 expression; shake flask culture;
D O I
10.1007/s11262-006-0061-0
中图分类号
Q3 [遗传学];
学科分类号
071007 ; 090102 ;
摘要
The partial VP2-encoding gene of Bluetongue virus serotype 23 (BTV-23) was amplified using reverse transcription polymerase chain reaction (RT-PCR) and inserted into pPICK9K vector. Recombinant plasmid DNA was integrated into the genome of Pichia pastoris by electroporation and expressed protein was identified by SDS-PAGE. High-level secreted expression was achieved after selecting the Mut(+) phenotype with multi-copy integrant in the recombinant yeast. The partial fragment of Bluetongue VP2 protein (BTV VP2) of approximately 45 KDa was secreted into the culture supernatant by the recombinant yeast when induced with methanol. Western and immuno dot-blotting methods confirmed the expressed BTV VP2 protein. The expressed protein has been demonstrated to be immunogenic in rabbits. A standardized method has been evolved for optimal expression and high-level production of the recombinant protein (284 mg/L). This is the first report demonstrating the possibility of mass production of BTV VP2 protein using P. pastoris.
引用
收藏
页码:265 / 271
页数:7
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