Abstract
An oligodeoxynucleotide library which the sequence were defined at 5 prime and randomized at 3 prime was employed to screen mRNA accessible targets,in reverse transcription and PCR after hybridized the library with mRNA. The mRNA of Glycophorin A(GPA),type I transmembrane glycoprotein,was screened and obtained 4 targets sequences. Accordingly 4 antisense nucleic acids designed respectively,the binding efficiency of every target were verified by using RNase H with antisense nucleic acids. Among them 2 targets showed better effects on binding and cutting. Designed 2 ribozymes to these targets,packaged in lentivirus system,then infected K562 cells(human erythroid leukemia line),the down-regulation effect of gene expression was validated by Real Time RT-PCR and by Western Blot. It was found that the screened targets showed the best effective knocking down effects on gene expression. The study provided a reference for mRNA targets screening and Ribozyme design,and was helpful in membrane receptor expression interference,since GPA is a transmembrane protein.
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