Construction and Expression of Vectors Expressing siRNA

The fragment of plasmid pCMV-tag-2B excluding Cytomegalovirus(CMV) promoter sequence was obtained by restriction enzyme digestion. A pair of primers was synthesized to amplify a fragment of the H1 promoter according to reference. The fragment of H1 promoter was amplified and inserted into the fragment of plasmid pCMV-tag-2B excluding CMV promoter sequence and plasmid pGEM- 11fz, respectively. The recombinant plasmids were named pCH1 and pGH1. A pair of primers, which included small interfering RNA(siRNA)target site for mRNA of EGFP gene, was synthesized and annealed in vitro, and then inserted into the corresponding vectors pCH1 and pGH1. In addition, the plasmid pEGFP-N3 expressing EGFP protein was transfected into liver tumor line Bel-7402 alone, or in combination with the plasmids producing siRNA to degrade mRNA of EGFP gene. The expression level of EGFP protein was viewed under the microscope. The results indicated that the plasmids including siRNA target site for mRNA of EGFP gene produced siRNA molecular and degraded mRNA of EGFP gene. The vector, PCH and pGH, will be used to study prevention of virus in fection.