[Expression and assembly of rotavirus-like particles in insect cells mediated by recombinant Bombyx mori MultiBac].
OBJECTIVE
To construct recombinant baculoviruses co-expressing three structural genes vp2, vp6 and vp7 of rotavirus, and assemble rotavirus-like particles (VLPs) in BmN cells.


METHODS
Human group A rotavirus was cultivated in MA104 cells, and the RNA was extracted and the three genes were obtained by RT-PCR. The PCR products were inserted into the transfer vectors pFBDM and pUCDM, respectively. A enhanced green fluorescent protein gene (egfp) driven by IE1 promoter was introduced into pFBDM to investigate the efficiency of infection. The expression baculoviruse was constructed by Tn7 and Cre-LoxP recombinant and transfected into BmN cells. The gene expression was determined by detecting 6-His tag fused into VP7 C-terminus, and the assembled VLPs were observed by transmission electron micrography.


RESULTS
Three genes of rotavirus were cloned and BmMultiBac was constructed. The genes were expressed and the rotavirus-like particles assembled in BmN cells successfully as verified by ELISA and electron microscope.


CONCLUSIONS
We have successfully constructed the recombinant baculovirus co-expressing the 3 structural genes of rotavirus, which provide the basis for producing protein complex containing multiple subunits and investigation of the structure of the macromolecules.
Long
Hu
H
College of Animal Science, South China Agricultural University, Guangzhou 510642, China. maclong@yahoo.cn
Yao
Lun-guang
LG
Wang
Shan-shan
SS
Chen
Shin-xin
SX
Tan
Pei-chan
PC
Sun
Jing-chen
JC
D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D012831:Bombyx / Q000821:virology; D036022:Capsid Proteins / Q000235:genetics*; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors; D012401:Rotavirus / Q000235:genetics* / Q000378:metabolism
D036022:Capsid Proteins