Essay --

The PCR products for each gene were purified using Qiagene purification kit. The T7 RNA polymerase gene was digested with NheI and XhoI. Then, after purification with a gel extraction kit (Qiagene), the DNA fragment of T7 RNA polymerase (in length of 2600Kb) was cloned into pIRES2-EGFP plasmid (clontech) and recombinant vector was called pIRES-T7. The cloning process for N and P genes were similar. The PCR products for each gene was purified and digested with NotI. The NotI site designed in 5’-end of reverse primers, but there was not any restriction enzyme site in forward primers. The forward primers contained a kozak consensus ribosme binding site (AACC) and ATG initiation codon. The pIRES2-EGFP plasmid was digested in a step by step process. First, pIRES2-EGFP was digested with BstxI and then, the digestion product of the plasmid treated by klenow to produce blunt end. Finally, pIRES2-EGFP was digested with NotI. The DNA fragments of N and P genes cloned into pIRES2-EGFP and recombinant vectors were called pIRES-N and pIRES-P, respectively. To produce tricistronic expression vecto...