The Pain Stimulation In Mice Under L Tetrahydropalmatine Induction And Inhibition Of Gene Screening

Objective:To construct a forward and reverse subjective cDNA library of genes transactivated by l-tetrahydropalmatine (l-THP) with the method of suppression subtractive hybridization in order to clone the genes , get them sequenced and analyzed,in which we expect to find the analgesia related target genes transactivated by l-THP which is needed for the further genetic engineering pharmaceutics. Method: the hot-plate test in ICR♀mice, mRNA was isolated from the brain of the two groups, and then cDNA was synthesized. In the forward hybridization, the induced genes screen was performed by using the l-THP group as Tester and the control group as probe(Driver); oppositely, the restrained genes screen was performed by using the control group as Tester and the l-THP group as Driver in the reverse hybridization. The gene fractions selected from the forward and reverse hybridization were used to set up the subtracted library. Amplification was carried out with E.Coli strain Top 10. The dot blot was used to screen the clones containing inserts. Then the selected clones were sequenced and analyzed in GeneBank with Blast search after PCR. Results: The forward amplified library contains 110 positive clones, the reverse amplified library contains 66 positive clones. Colony PCR show 35 forward and 15 reverse clones containing inserts, 6 and 5 clones of each were selected after the dot blot screen. Sequence analysis was performed also.It was found that 3 kinds of known cDNA sequences may be the target genes transactivated by l-THP. Conclusion: The subtracted library of genes transactivated by l-THP was constructed successfully. It provides a foundation for the further genic mechanism research of l-THP.