The Construction Of COX-2 Over-expression Vector And Which Express In Hscs

Objective: To build cox-2 expression vector and observe its expression in transfecting HSC-T6, for further studying the relation between cox-2 over-expression and the biological functions of HSC.Method: 1. To abstract the COX-2 murine gene and then obtain the rat liver m RNA by the method of TRIzol, after all of this, reversing transcribed into the establishment of c DNA Library. 2. To obtain the target fragment of COX-2 by RT-PCR amplified, after the Double enzyme digestion of PCR product and pc DNA3.1(+) Plasmids, connecting the Gene fragment of COX-2 to the pc DNA3.1(+) vector, transferring the constructed pc DNA3.1(+)-r COX-2 to E. coli. DH5α competent cells, and selecting the positive clone strains in 50 g/ml Kana LB solid plate. 3. After selection, to analyze the double enzyme digestion, agarose gel electrophoresis and sequencing. 4. To transfect the successful construction of pc DNA3.1(+)-r COX-2 expression vector into HSC-T6 cells through Liposome, and then setting up the experimental groups: The group of HSC-T6 cells(control group), the group of pc DNA3.1(+)-r COX-2-1(over-expression group); the group of pc DNA3.1(+)(empty vector). 48 hours after transfection, testing the expression of COX-2 m RNA which in the HSC-T6.Results:1. The RT-PCR products show that: the band appeared in about 1800 bp.2. The target gene enzyme digestion, Sequence analysis and the sequencing results show that it constent with the COX-2 gene sequence of Gen Bank, which means the pc DNA3.1(+)- r COX-2 expression vector was successfully constructed. 3. After the pc DNA3.1(+)-r COX-2 expression vector transfected into HSC-T6 cells, it appears that: the COX-2 m RNA in the group of pc DNA3.1(+)- r COX-2-1 was significantly higher than which is in the control group and empty vector group, It is a significant in Statistical processing(p<0.01).Conclusions: 1. The construction of rat COX-2 expression vector is successful. 2. The constructed pc DNA3.1(+)-r COX-2 expression vector could get the higher expression which provides preparatory for further studying the relation between cox-2 over-expression and the biological functions of HSC.