Cloning of Rota Virus Outer Capsid Protein (VP7) Gene into the pGEM Vector

Abstract

Background and Aims: In humans the group A 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 and virtually eliminating hospitalizations due to rotavirus gastroenteritis, a heavy burden in developed countries. A reassortant Rhesus-human rotavirus vaccine, Rotashield was manufactured and marketed in 1998. Nine months later a small number of cases of intussusceptions were identified, leading to the withdrawal of vaccine. Important questions remain for these oral live vaccines related with safety and side effects as well as production costs, thus development of second generation of nonreplicating rotavirus vaccine should be considered as an alternative to live vaccines. VP7 is major outer capsid glycoprotein; can induce production of neutralizing antibodies. Regarding these concepts we cloned VP7 gene of SA11 rotavirus in pGEM vector for future expression. Methods: With large scale expression and purification of VP7 protein, it will be convenient to study VP7 antigenic, immunogenic, biologic functions and its potential for vaccine development. Results: BSC-1 cells were grown as a monolayer. Simian rotavirus, SA11 was cultured. Oligonucleotide forward and reverse primers, specific for gene segment 9 which encodes VP7, were synthesized, according to dsRNA gene segment 9 of simian rotavirus SA11 sequence data at NCBI GeneBank. Rotavirus ds-RNA was extracted and used as template for reverse transcription to synthesize cDNA from both viral strands. cDNA product of RT-PCR was cloned into pGEM@-5Zf(-) vector and the results showed that rotavirus genome segment 9 was cloned into pGEM@-5Zf(-) vector and confirmed by restriction enzyme and sequencing. Conclusion: Sequencing result was analyzed by BLAST software showing 100% homology with 9 1 SA11 rotavirus genome segment in NCBI GeneBank.