Recombinant outer capsid glycoprotein [VP7] of rotavirus expressed in insect cells induces neutralizing antibodies in rabbits

Rotaviruses cause diarrhea in infants and young children worldwide. Rotavirus outer capsid protein, VP7 is major neutralizing antigen that is important component of subunit vaccine to prevent rotavirus infection. Many efforts have been done to produce recombinant VP7 that maintain native characteristics. We used baculovirus expression system to produce rotavirus VP7 protein and to study its immunogenicity. Simian rotavirus SA11 full-length VP7 ORF was cloned into a cloning plasmid and then the cloned gene was inserted into the linear DNA of baculovirus Autographa californica Nuclear Polyhedrosis Virus [AcNPV] downstream of the polyhedrin promoter by in vitro recombination reactions. The expressed VP7 in the insect cells was recognized by rabbit hyperimmune serum raised against SA11 rotavirus by Immunofluorescence and western blotting assays. Rabbits were immunized subcutaneously by cell extracts expressing VP7 protein. Reactivity with anti-rotavirus antibody suggested that expressed VP7 protein had native antigenic determinants. Injection of recombinant VP7 in rabbits elicited the production of serum antibodies, which were able to recognize VP7 protein from SA11 rotavirus by Western blotting test and neutralized SA11 rotavirus in cell culture. Recombinant outer capsid glycoprotein [VP7] of rotavirus expressed in insect cells induces neutralizing antibodies in rabbits and may be a candidate of rotavirus vaccine

Decolorization of different azo dyes by Phanerochaete chrysosporium RP78 under optimal condition

Detoxification of synthetic dyes is one of the main challenges in clearing textile industry wastes. Biodegradation of azo-dyes using Phanerochaete chrysosporium is one the most environmentally friendly methods available. The main enzymes responsible for mycodecolorization process are lignin and manganese peroxidases. Here, optimization of expression conditions has been carried out with manipulating culture condition and nutrient sources. Therefore, the effects of buffer and temperature as well as nitrogen source on lignin peroxidase and manganese peroxidase production were investigated at two levels and four levels, respectively. For this purpose, P. chrysosporium RP78 based on Taguchi design of experiment has been applied. Maximum lignin and manganese peroxidase activities of 182 +/- 2.5 U/L and 850 +/- 41 U/L were obtained under predicted optimum conditions, respectively. Thereby, about 100% decolorization was achieved after 24 h for two most widely used groups of azo dyes in textile industry consisting reactive and acidic. The physical adsorption of the azo dyes by mycelia was not significant which indicated that the enzymatic degradation of the dyes was occurred. Time profile of these enzymes showed that manganese peroxidase was peaked on 9 th day while lignin peroxidase peaked on 13 th. day and remained stable in the culture. The extracellular expression profiles of both were studied by 2 dimensional gel electrophoresis to partially characterize the enzymes

[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 Gene Bank. Sequencing result confirms highly that the nucleotide sequences of VP4 gene after long and continuous passage of SA11 rotavirus is conserved in our laboratory