Construction, Expression And Identification Of G Protein Polypeptide Of Human Respiratory Syncytial Virus Expressed By Recombinant Adenoviruses

Objective: Synthetic G-protein polypeptide comprises two disulfide bridges and multiple protective B cell epitopes. The G-protein polypeptide which immuned the animal models generated a highly protective antibody response against RSV challenge . Therefore, the G-protein polypeptide represents a new potential antigen for an RSV vaccine. We used the replication-defective adenovirus vector and the homologous recombination , so we obtained the recombinant adenovirus by carrying the G-protein polypeptide .Methods: Based on the base sequence of the G-protein polypeptide, the oligonucleotide primers were designed and synthesized which contains the mutational nucleotide sequence. The G-protein polypeptide DNA was obtained by the PCR mutation which the template was the RT-PCR production of G protein and the pfu DNA polymerse was used. So the Taq DNA polymerse added the dATP to the PCR product 3' end of the G-protein polypeptide with pGEM-T easy vector. The gene of the G-protein polypeptide was cloned into the shuttle vector pShuttle-CMV, thus the recombinant shuttle plasmids were obtained. The resultant constructs were linearized with Pme I and transformed along with the supercoiled adenoviral vector pAdEasy-1 into RecA~+ E.coli strain BJ5183.The recombinant adenoviral constructs were cleaved with Pac I to expose their inverted terminal repeats (ITR) and transfected into 293 packaging cells to generate viruses. The recombinant adenovirus containing the G-protein polypeptide was obtained. The G-protein polypeptide specific DNA was integrated in the viral genome being identified by PCR assay and the viral genomes was stable when tested by PCR...