ABSTRACT
<p><b>OBJECTIVE</b>To evaluate the effectiveness of small interfering RNA (siRNA) on inhibiting severe acute respiratory syndrome (SARS)-associated coronavirus replication, and to lay bases for the future clinical application of siRNA for the treatment of viral infectious diseases.</p><p><b>METHODS</b>Vero-E6 cells was transfected with siRNA before SARS virus infection, and the effectiveness of siRNA interference was evaluated by observing the cytopathic effect (CPE) on Vero-E6 cells.</p><p><b>RESULTS</b>Five pairs of siRNA showed ability to reduce CPE dose dependently, and two of them had the best effect.</p><p><b>CONCLUSION</b>siRNA may be effective in inhibiting SARS-associated coronavirus replication.</p>
Subject(s)
Animals , Chlorocebus aethiops , RNA, Small Interfering , Pharmacology , Severe acute respiratory syndrome-related coronavirus , Transfection , Vero Cells , Virus ReplicationABSTRACT
AIM: To construct E. coli expression plasmid of recombinant human NDPK-A with a 6?His tag, optimize the expression condition and identify the activity of the product. METHODS: nm23-H1 was subcloned from plasmid pBVNMH1 to pQE40 which contain 6?His purification tag. The expression condition was modulated in grades to get the optimal expression. We purified protein with the Ni+-NTA affinity chromatography column, identified the immunogenicity of the product with Western blot, and measured the kinases activity with HPLC. In addition, angiogenesis inhibition activity of rhNDPK was identified by CAM. RESULTS: The sequence of nm23-H1 subclone in pQE40 was exactly correct. The expression rate of rhNDPK-A was 49 6%. Purified rhNDPK-A specially recognized the antiserum of NDPK-A. It also inhibited angiogenesis. CONCLUSION: PQE-nm23H1 containing 6?His can express target protein at high level. This purification method is simple than other methods, and the product has the same activity as natural human NDPK-A.