Fig. 1.

Schematic diagram for the construction of the chimeric S gene and production of a full-length cDNA of rH120-S1/YZ. (A) Replacement of the H120 S1 fragment by the corresponding sequence of IBYZ for construction of pYZS1H120S2. The plasmids pIBYZS and pH120S contained the S gene of IBYZ strain and H120 strain, respectively, were constructed during the establishment of reverse genetic system. Primers PS1F and PS1R were used to amplify the S1 fragment of IBYZ and vector fragment, while primers PS2F and PS2R were used to amplify the S2 fragment of H120 strain. By overlapping PCR, the pYZS1H120S2 was constructed which contained a chimeric S gene. (B) Strategy for the construction of full-length cDNA clones of rH120-S1/YZ. Ten cDNA fragments covering the entire genome of H120 strain was amplified by RT-PCR. Unique Bsa I sites were inserted at the junctions between each clone, a unique T7 start site was inserted at the 5′ end of clone TM1, and a 28-nucleotide T tail was inserted at the 3′ end of clone TM10. The S fragment in the pMDTM8 plasmid of H120 strain was replaced by chimeric S gene. By using appropriate ligation strategy, the genomic cDNA of rH120-S1/YZ was assembled by in vitro ligation using appropriate restriction sites as indicated.