Cloning, Expression And Characterization Of Vaccinia Growth Factor-a Virokin Encoded By The Tian Tan Strain Of Vaccinia Virus

Vaccinia growth factor (VGF), a new member of EGF superfamily, is capable of binding or stimulating EGF receptor autophosphorylation to a level greater than that of human EGF. The development of recombinant VGF for the treatment of nonhealing or badly healing wounds and for the detection of EGF receptor in sample has been highly desired. VGF is encoded by an early gene (originally designated 19K), and the primary translation product is 140 amino acids long. The glycosylated transmembrane precursor is cleaved in two locations to release a signal sequence of 19 amino acids (from residues 1 to 19) , a mature VGF of 77amino acids (from residues 20-96), and to remove the transmembrane sequence and its cytoplasmic domain .3 primers were synthesized. V3 was used as 3' primer, while V5n and V5m (both containing Bgl II site) as 5' primer. From the Tian Tan strain of vaccinia virus, the gene encoding mature VGF (from residues 20 to 96, named VGFm) was amplified by PCR with V3/V5m primer for prokaryotic expression and the other encoding peptide from residues 1 to 96 (named VGFn) with V3/V5n primer for eukaryotic expression. The recombinant plasmid pSP72/VGFm and pSP72/VGFn were constructed by cloning digested PCR products to pSP72 plasmid digested with EcoR I...