[Expression of capsid gene of Chinese isolate of rabbit hemorrhagic disease virus in Pichia pastoris].
The capsid protein (VP60) gene of RHDV was subcloned into the Pichia expressin vector pPICZ B to express the VP60 protein intracellularly. The recombinant plasmid was initially transformed into a E. coli strain TOP10 F'. After verification of the construct by sequencing, the recombinant plasmid was linearized by Sac I in the 5' AOX1 region and then transformed into Pichia pastoris strain GS115 using the Pichia EasyComp Kit. After selecting and verifing for the insertion of VP60 gene in the genome, two clones of Pichia transformants were select for expression test. The recombinant clones were first inoculate with BMGY in baffled flask at 28-30 degrees C in a shaking incubator (250-300 r/min) until culture reaches an OD600 = 2-6, then resuspend the cell pellet to an OD6oo of 1.0 in BMMY medium to induce expression for 5 days by methanol at a concentration of 0.5% in a 1 liter baffled flask covered with 2 layers of sterile gauze. Collect the cell pellets and break it by acid-washed 0.5 mm glass beads. The expression of recombinant Pichia strains was detected by SDS-PAGE and Western analysis with a polyclonal serum which showed a specific protein band of 60kD. Theses results indicates that the recombinant VP60 produced in Pichia was antigenically similar to the viral polypeptide. Electron microscopic observation of the recombinant Pichia-derived protein revealed the presence of virus-like particles similar in size and appearance to native virus capsids. In the haemagglutination test, the recombinant VLPs, like the native RHDV, also agglutinated human blood type O erythrocytes and could be inhibited by the anti-RHDV polyclonal serum.
Yan
Wei-Wei
WW
College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China.
Cui
Zhi-Zhong
ZZ
Wang
Yong-Kun
YK
D000818:Animals; D002213:Capsid / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D017929:Hemorrhagic Disease Virus, Rabbit / Q000235:genetics*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D015678:Viral Structural Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D015678:Viral Structural Proteins; C081149:viral protein 60, rabbit hemorrhagic disease virus