Construction And Detection Of Rna Interference Vectors For Silencing Over-expression Vector And Rnai Expression Vector Of α-1,3Galactosyltransferase Gene From Guangxi Bama-mini Pig

Alpha-1,3-galactosyltransferase gene is also known as the GGTA1gene. This gene codes for the enzyme α-1,3galactose transferase, which catalysis α-Gal epitope on the cell surface of most mammals, but not on those of humans and Old World monkeys. Because the lack of GGTA1gene expression, no α-Gal epitope can be detected on the surface of human cells, therefore large amounts of a natural antibody designatesd ’anti-α-Gal’ were produced, which is thought to be the major immediate barrier to successful clinical Xenotrans-plantation due to hyperacute rejection occurring. In the present study, RNAi vectors and over-expression vector for Bama mini-pig’s GGTA1gene were constructed, and silencing efficiencies in cells were evaluated. This research will be helpful to constitute an important foundation for breeding GGTA1gene silencing Bama mini-pig’s which can be used in xenotransplantation research.A pair of primers added with both HindⅢ and BamH Ⅰ restriction sites which were synthesised according the GGTA1gene sequence released in NCBI, and the cDNA is amplified by RT-PCR using the total RNA from the liver tissue of Bama-mini pig as templates. The amplified segments were ligated into pMD18-T vector after purification and then the recombined vector was transformed into E.coli DH5a for sequencing. Two cDNA segments including one is losing the total exons of GGTA1designated as GGTA1-1and another segments contain full length of GGTA1designated as GGTA1-2were amplified. The two cloned segments were then inserted, respectively, to the pDsRed1-N1plasmid, and constructed two recombined eukaryotic plasmid which named as pDsRed1-GGTA1-1and pDsRed1-GGTA1-2. Finally, the recombined plasmid was transfected into the PK-15cells mediated by Lipofectamine2000. After24hours transfection, the expression of the exogenous gene in cells was detected by inverted microscope. The results showed that the pDsRedl-GGTA1-1and pDsRed1-GGTA1-2recombined plasmid can expression in the PK-15cells.In order to evaluate the RNAi silencing efficiency on GGTA1expression, three RNAi vectors including pLLU2G-shGGTA1-1、pLLU2G-shGGTA1-2、 pLLU2G-shGGTA1-3, and a negative control vector named as pLLU2G-shGGTA1-control were constructed. Western blot and QRT-PCR was employed to detect the silencing efficiencies of GGTA1in PK-15cells after transfected into the constructed vectors as above mediating with the Lipofectamine. The results of Real-time PCR showed that the silencing efficiencies of pLLU2G-shGGTA1-1, pLLU2G-shGGTA1-2, pLLU2G-shGGTA1-3were85.02%,86.05%,83.85%, respectively. The pLLU2G-shGGTA1-Control and two blank control group including lipofectamine2000and DMEM had no silencing effect. The GGTA1gene expression levels of positive control (pDsRed1-GGTA1-1vector) far higher than the blank group. The results of Western blot showed that cells transfected with the three silencing vors only expressed trace almost of GGTA1protein, which in accordance with that obtained by real-time PCR. The results from real-time PCR and western blot indicated that there was not GGTA1expression difference between the cells transfected with pLLU2G-shGGTA1-Control group, lipofectamine2000and DMEM (blank control group). However, the cells transfected with pDsRedl-GGTA1-1vector (positive control) showed markedly higher GGTA1protein expression levels.In conclusions, the three interference shRNA in the study can down-regulate the expression of GGTA1gene, of which the down-regulation effici-ency of shGGTA1-2the highest as to86.05%, indicating that it can be used to silencing GGTA1gene, to develop GGTA1gene expression silencing animal model in xenotransplantation research.