[Construction and efficacy study of a novel 10-23 deoxyribozyme expression vector].
OBJECTIVE
To construct a novel 10-23 deoxyribozyme (10-23DZ) expression vector, identify the intracellular production of specific 10-23DZ and its inhibitory effect upon the expression of TACO gene in macrophage.


METHODS
An oligonucleotide containing the primer binding sequence of mouse Moloney leukemia viral reverse transcriptase (MLV-RT), a multiple cloning site (MCS) and a stem-loop structure for termination of reverse transcription was designed and inserted into one MCS of plasmid pBUDCE4.1, downstream of the cytomegalovirus promoter (P(CMV)). The gene fragment encoding MLV-RT was inserted into another MCS of pBUDCE4.1, downstream of elongation factor 1alpha promoter (P(EF-1alpha)). The resulting plasmid was named pSDE01. Then pSDE01 was transfected into RAW264.7 cell and the expression of MLV-RT and the reverse transcriptase activity of expression product was identified by RT-PCR. To identify the cellular expression of 10-23DZ by pSDE01, a 10-23DZ targeting the TACO mRNA of macrophage was designed according to the predicted secondary structure of TACO mRNA. The expression sequence of designed 10-23DZ, DZ1, was synthesized and inserted into the MCS of ODN-PSL in pSDE01. The resulting plasmid, pSDE01-DZ1, was transfected into RAW264.7 cell and the expression of DZ1 was identified by PCR and dot-blot respectively. At 48 h after transfection of pSDE01 into RAW264.7 cells, total RNA and proteins were extracted and the TACO mRNA and protein expression level was assayed by RT-PCR and Western blotting respectively.


RESULTS
Restrictive analysis and sequencing data showed that the 10-23DZ expression vector, pSDE01, was successfully constructed and could express MLV-RT with reverse transcriptase activity in cell. When transfected into RAW264.7 cells, pSDE01-DZ1 expressed DZ1 effectively and inhibited the expression of TACO gene. And TACO mRNA decreased by 77.7% (0.193 +/- 0.008 vs 0.864 +/- 0.005, P < 0.05) and TACO protein decreased by 73.3% (0.114 +/- 0.051 vs 0.427 +/- 0.043, P < 0.05).


CONCLUSIONS
A novel expression vector capable of generating specific 10-23DZ in cells has been successfully constructed. And the intracellular product 10-23DZ may inhibit the expression of any target gene of interest.
Li
Jun-Ming
JM
Department of Clinical Laboratory, First Affiliated Hospital of Nanchang University, Nanchang 330006, China. lisir361@163.com
Wan
La-Gen
LG
Zhang
Cai-Cheng
CC
Wang
Na
N
Luo
Qing
Q
D000818:Animals; D002460:Cell Line; D021881:DNA, Catalytic / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D008264:Macrophages / Q000378:metabolism; D008840:Microfilament Proteins / Q000235:genetics; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection
D021881:DNA, Catalytic; D004277:DNA, Single-Stranded; D008840:Microfilament Proteins; D012333:RNA, Messenger; C402582:RNA-cleaving DNA 10-23