Construction And Expression Of Lentiviral Vectors Enconding Recombinant Goat Grp78 In Eec Cells

Endoplasmic reticulum(ER) is one of the important cellular organelles responsible for protein processing and calcium storage. The accumulation of misfolded and/or unfolded proteins and/or calcium dyshomeostasis in ER induces stress, which would in turn enhance the cell resistance and adaptability to injuries through the unfolded protein response pathway.Glucose-regulated protein 78(GRP78), also referred to as BiP/HSPA 5, is a central regulator of endoplasmic reticulum function and induction of GRP78 has been widely used as a marker for endoplasmic reticulum stress and the onset of the unfolded protein response.According to the goat in the NCBI GRP78 gene sequences, with construction pCD513 B lentivirus vectors contain full length cDNA of GRP78 expression vector, use lentivirus vectors interference pCD513B-U6 build for GRP78 gene sequences of shRNA interference and to interfere with control the interference of meaningless sequence carrier.The shuttle plasmid and packaging plasmid transfection HEK 293 cells for lentivirus packaging.Will the virus infection after suspension gradient dilution HEK 293 cells to detect virus drops.The established stable clones could routinely producevector supernatants with titers above 107 transduction units per milliliter(IU/mL).Then,endometrial epithelial cells(EEC) cells with lentivirus infection.Using RT-PCR and western blotting method to detect the expression of GRP78.1. The goat Grp78 has an open reading frame of bp coding sequences which encoded amino acids. From the deducrd amino acid sequence, the molecular weight(Mw) and the oretical isoelectric point(pI) of the goat Grp78 were 72397.0 u and 5.07, respectively.2. Grp78 gene was connected in to pCD513 B vector and renamed the expression vector pCD513B-Grp78. The result of agarose gel electrophoresis of recombinant pCD513B-Grp78 digested by restrictive endonuclease showed special bands of 8100 bp and 2300 bp. It confirmed that the pCD513B-Grp78 was constructed successfully.3. Five Pairs of small hairpin oligonucleotide targeting ORF of Grp78 gene were designed and synthesized. The oligonueleotides were annealed to produce complementary double-strand as siRNA gene and then cloned into the lentiviral-vector pCD513B-U6-shRNA-Grp78. The 2.0% agarose gel electrophoresis results of PCR showed the 350 bp gene fragment was obtained by amplifying with a pair of specialprimers designed. It confirmed that the pCD513B-U6-shRNA-Grp78 was constructed successfully.4. The correct recombinant lentiviral-vector plasmid and packing mixer were co-transfeeted into HEK 293 cells, then recombinant lentiviruses were produced.5. EEC cells infected with lentiviral vector carrying full-length Grp78 gene successfully expressed high-level Grp78. The mRNA of Grp78 reduced to less than 70% after delivery of lentiviral vector carrying shRNA sequence.The lentiviral vectors carrying the full length Grp78 gene and shRNA sequence targeting Grp78 have been successfully constructed.It has laid an experimental basis on its functional study.