فهرست مطالب مرتضی تقی زاده طرنابی

Influenza virus, which is a decisive agent of influenza, attracted interest because of the event of the annual epidemic among a wide range of animal and human hosts. This study aimed to express the hemagglutinin protein of Iranian swine Influenza A (H1N1) through a baculovirus system in SF9 cells to produce a new recombinant vaccine.

Hemagglutinin gene of Swine H1N1 virus was amplified with specific primers containing restriction enzymes site, and then cloned. Afterward, the vector was transformed to DH10Bac using Bac-to-Bac system in order to produce a recombinant bacmid. Hemagglutinin expression and its biological activity were assessed using molecular and immunization tests.

The target rHA in length, 1710 bp, was produced and expressed in transfected SF9 cells with a size of ~66 kDa. The infected cells expanded in size and their nucleus, and desiccated from the surface of the cell culture as a granular. They could absorb chick red blood cells (RBCs), and appear as cell aggregates forty-eight postinfection. The fact that infected cells were unable to form cell clamp showed the test's accuracy and inhibition of hemodesorption activity. The amount of protein obtained was 10.76 µg/100 µl, equal to 0.1 mg/ml.

The baculovirus expression system could express the recombinant protein in the insect cell. Therefore, it may be a well-suited alternative to produce a new generation of the vaccine instead of egg-based and cell-culturebased generation vaccines.

According to marked advances in bioinformatics studies, development of influenza vaccines has been greatly modified in many studies. In this study, we have designed a multi-epitope recombinant protein, consisting of several conserved epitopes from Hemagglutinin protein of the H1N1 and H5N1 strains of Influenza virus by immunoinformatics approaches.

The registered sequences of hemagglutinin proteins from H1N1 and H5N1 strains have been analyzed and selected epitopes have been chosen and directed for further analysis. After investigation of immunological and physicochemical parameters, the selected regions were fused together by linkers and sequence of target constructs was determined following codon optimization. The target construct has been integrated into BamHI/XhoI cleavage sites of PET32a+ expression vector.

A physicochemical analysis revealed that the designed recombinant protein has 214 amino acid with molecular weight of 22.451 kDa. The isoelectric point was determined as 7.25 , solubility of 100% and  Extinction coefficient of 60170 M-1cm-1.finally, after codon optimization, the Codon Adaptation Index (CAI) was calculated as 0.95.

According to these results, it has been predicted that designed multiepitop recombinant protein has reasonable and unique immunological properties. The expressed protein could be used for future immunological studies against type A influenza virus