Cloning andd expression of Fusion gene of measles virus (AIK-C strain)
2007
Aqa`i Pur, Khosru | Shafi`i, A`bbas | Safaviyeh, Sediqeh Sadat | Esma`il Zadeh, Majid
Measles virus is classified as a member of Morbilivirus genus in the family of Paramyxoviridae. Two glycoproteins of this virus, H and F are important in anti MV immune response. In recent years, a number of experimental vaccines have been developed which could [ass the problem of neutralization of the live measles virus vaccine by maternal antibodies. In this research full length of Fgene was isolated, analysed and cloned in pET15b plasmid. MRC-5 and Vero cells vere cultivated and infected with measles (AIK-C). Total RNA seprated and purified with acid-phenol-guanidium method, from infected cells. Specific cDNA for fusion protein was synthesized by specific primers. Then the cDNA amplified and ligated into plasmid. The ligation product was transformed into DH5ل (E.coli strain). The transformed bacteria screened with ampicilin reseistance gene and PCR. Clonies with positive PCR were grown in LB broth containing 100 ىg/ml ampicilin. The plasmids were isolated and purified with alkaline lysis and their sizes were analyzed on agarose gel electrophoresis. Recombinant plasmids were evaluated by PCR and digestion with restriction enzymes. Nucleotide sequences were determined by dedeoxy chain termination method in MWG Biotech Company. The recombinant plasmid were expressed in BL21 (E. coli strain) and the recombinant protein were visulalized on SDS-PAGE by commasie blue and silver staining.
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