Vaccine potential of Rotavirus Outer Capsid Protein (VP4) cloned into the plasmid

Rotaviruses are associated with endemic diarrhea in children under the age of 5, leading to approximately 800,000 deaths every year. Introduction of rotavirus vaccines into childhood immunization programs can contribute to substantial reduction in mortality from rotavirus gastroenteritis in developing countries. VP4 is outer capsid spike protein of rotavirus by which virus can bind to its receptor. VP4 protein can induce production of neutralizing antibodies. Regarding these concepts we cloned VP4 gene of rotavirus in plasmid vector for future expression. BSC-1 cells were cultured as a monolayer. Rotavirus CPE positive cell cultures were freeze-thawed three times. Rotavirus was partially purified by ultracentrifuge. Oligonucleotide primers, specific for gene segment 4 which encodes VP4, were synthesized. Rotavirus RNA extracted and used as a template for synthesis of cDNA by reverse transcriptase. Then they proliferated by PCR and PCR products were cloned into plasmid vector which was analyzed by restriction enzymes and sequencing. Sequencing result was analyzed with BLAST software that had a 100% homology with SA11 rotavirus genome segment 4 in NCBI GeneBank. Sequencing result confirms highly that the nucleotide sequences of VP4 gene after long and continuous passage of SA11 rotavirus is conserved in our laboratory.