[Characterization and secreted expression of dengue virus type I-IV envelope glycoprotein domain III in Pichia pastoris].
OBJECTIVE
To achieve secretory and extracellular production of recombinant dengue virus serotypes I-IV envelope glycoprotein domain III (DENV-1-4 EDIII) in Pichia pastoris.


METHODS
EDIII genes of DENVI-IV were amplified and cloned into vector pPIC9K, respectively. These recombinant plasmids were then linearized and transferred into Pichia pastoris strain GS115. Clones highly produced in 4.0 mg/ml G418 were amplified and induced by methanol to achieve the secreted recombinant proteins. Ni-NTA agarose beads were used for purification, while SDS-PAGE and Western blotting were used for identification.


RESULTS
The recombinant plasmids pPIC9K-DENV-1-4 EDIII were constructed and successfully transferred into Pichia pastoris strain GS115. The recombinant EDIII proteins were expressed in a secretory way with the molecular weight about 12 × 10(3) and specifically identified by anti-His monoclonal antibody and anti-DENVI-IV mice sera.


CONCLUSIONS
DENVI-IV EDIII proteins are successfully achieved from Pichia pastoris expression system and could be used for development of dengue vaccines, diagnostic reagents and study of biological function of the E protein.
Clinical Laboratory, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.
Jian-piao
JP
Cai
Fei
F
Qian
Jia-ying
JY
Wang
Ying
Y
Zhao
Xiao-jing
XJ
Xu
Wei-rong
WR
Jin
Xiao-yan
XY
Che
D003716:Dengue Virus / Q000235:genetics*; D005822:Genetic Vectors; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics; D014759:Viral Envelope Proteins / Q000378:metabolism*
D011994:Recombinant Proteins; D014759:Viral Envelope Proteins