Cloning and expression of simian rotavirus spike protein (VP4) in insect cells by baculovirus expression system.
VP4 protein is as spikes on rotavirus outer capsid shell which is responsible for virus attachment to the host. VP4 induces production of neutralizing antibodies which could be used for serotyping of different isolates.



Simian rotavirus SA11 gene 4 cDNA was cloned into a cloning plasmid pDONRTM by recombination reaction using clonase II enzyme mix. The resulting clone was called VP4-entry clone. In the second recombination reaction, cloned gene was inserted into the linear DNA of the Baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) downstream of the strong polyhedrin promoter. The recombinant AcNPV-VP4 DNA was transfected by lipofection into the insect cell line, Spodoptera frugiperda (Sf9) cells. Expression of VP4 in the Sf9 cells was confirmed by the immunofluorescence test using rabbit polyclonal anti-rotavirus and anti-rabbit FTIC-conjugated antibodies by Western immuno-blotting technique. The antigenicity of the expressed protein was determined by immunizing rabbits and testing the sera by Western-blotting and neutralization method.



The cloned VP4 gene was obtained and expressed in baculovirus system. The specificity of the expressed protein was confirmed by its reactivity with anti-rotavirus antibody. Antibody produced against the expressed protein showed neutralizing activity for rotavirus indicating that the protein was biologically active and could induce natural antibody response.



The expressed protein from rotavirus VP4 gene has a potential for development of rotavirus vaccine.
Khodabandehloo
Mazaher
M
Dept. of Virology, Iran University of Medical Sciences, Rassul Akram Hospital, Tehran, Iran.
Shamsi Shahrabadi
Mahmoud
M
Dept. of Virology, Iran University of Medical Sciences, Rassul Akram Hospital, Tehran, Iran.
Keyvani
Hossein
H
Dept. of Virology, Iran University of Medical Sciences, Rassul Akram Hospital, Tehran, Iran.
Bambai
Bijan
B
National Institute for Genetic Engineering and Biotechnology, Pazhoohesh Boulevard, Kilometrs17, Karaj Highway, P.O. Box 14455-6343, Tehran, Iran.
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D004587:Electrophoresis, Agar Gel; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D007313:Insecta / Q000166:cytology*; D007700:Kinetics; D012313:RNA / Q000032:analysis; D012401:Rotavirus / Q000235:genetics*
D036022:Capsid Proteins; C060339:VP4 protein, Rotavirus; D012313:RNA