CDNA Cloning And Expression Effects Of Porcine Growth Hormone Gene

A cDNA for porcine growth hormone (pGH) was amplified by RT-PCR using total RNA from porcine pituitary as the template. The primer pairs included: the sense primer with the sequence as 5'-CTAGGATCCCCGGCTGTGATGGCT GCA-3' (BamHI) and the aitisense primer with the sequences as 5'-CAGAATTC GCAACAGAGATGCCCAG-3' (EcoRI). The pGH cDNA ORF obtained encodes a GH peptide precursor with 216 AA. The protein sequence encoded by the cDNA had 100% homology to that of Dahe pig and shares 98.6% - 99.5% homologies to the GH precursors from other local pig breeds.The prokaryotic expression and antibody preparation of pGH: This cDNA was correctly inserted into pGEX4T-1 vector in order to construct the prokaryotic expression plasmid pGEX4T-1/pGH. A new protein band with molecular weight of 50.4 KDa was found through the analysis of SDS-PAGE on the induced expression product of pGH. This proved that the recombinant pGH has been expressed.The fusion protein GST-pGH expressed in the form of inclusion bodies was isolated; the recombinant fusion protein that recovered from polyacrylamid gel was used to immunize rabbits. The specific polyclonal antibody for pGH was prepared and purified. The anti-pGH serum from immunized rabbits was proved specialized against the pGH through the detection of ELISA (Enzyme Linked Immunosorbent Assay), Western Blot Analysis and Immunohistochemical detection. The polyclonal antibody for rabbit anti-pGH purified by caprylic acid-ammonium sulfate method reached the purity by SDS-PAGE. Purified polyclonal antibody can specifically bind with the fusion protein of the recombination pGH (about 50.4 KDa) and the natural pGH (24.4 KDa) strived from pig pituitary. The purified antibody can check positive immune signal of the prepituitary gland cell by immnuno- histochemistry analysis.The expression of pGH cDNA in yeast was also studied by constructing the expression plasmid pPIC3.5K-pGH. The cDNA coded fragments area was orientedly inserted into the expression plasmid pMD18-T. The recombinant plasmid was transformed into E.coli JM109 competent cells; the positive clone was screened by colony PCR and plasmid PCR; the recombinant plasmid was identified by BamH I and EcoRI digestion, and digested by BamH I and EcoRI. The goal enzymes obtained were directly inserted into pPIC3.5k plasmid, which was named the recombinant plasmid pPIC3.5K-pGH. After linearly cleavage of Sal I digestion, the pPIC3.5K-pGH plasmid was transform into GS115 yeast by lightning stroke, then MD and MM mediums were used to screen the recombinants. These recombinant cells were cracked and the genome DNA was extracted. PCR was used to authenticate them being correct recombinants with pGH. The recombinant pGH in yeast was expressed by the induction of methanol and the protein product was analyzed by SDS-PAGE and Western-blotting. The recombinant yeasts expressed high effectively by the induction and screening was named pPIC3.5K-pGH/GS115. SDS-PAGE and Western-blotting proved that the recombinant yeast had transcripted pGH whose molecular weight was close to 25 KDa. This size is consistent with the expectation of 24.4 Kda of the protein coded by pGH gene.Expression of pGH in mammal cells: cDNA of porcine growth hormone (pGH) was also correctly inserted into a eukaryotic expression vector VR1020 and resulted in the expression plasmid VpGH. The VpGH plasmids wrapped with thin film liposome was transfected into COS₇ cells. Total RNA of the transfected cells was extracted at 24 h, 48,h and 72 h respectively. RT-PCR, ELISA and Immuno-fluorescence assay were used to analysis the COS₇ cells transfected. RT-PCR results showed the mRNA from corresponding cDNA was found in the total RNA of the transfected cells within 24 h; the expressed protein product of pGH gene was found in the COS₇ cells transfacted, basing on ELISA and Immuno- fluorescence assay. We also used VpGH wrapped with liposome to transfecte animals (mice and pig) in vivo in order to identify the effects of the preparation of VpGH/liposome on growth. Kunming mice were injected with VpGH, VR1020 and NaCl solution separatively. Their effects were analyzed on the body weight, the number of immune cells, and the content of GH in mice serum and the immune level of mice body liquid. The results showed that the new preparations of VpGH wrapped with liposome could promote the growth and increase the number of white blood cells of these mice significantly, but not improve the immune level of mice body liquid.A similar experiment was carried out in the pigs. We injected the pigs with VpGH wrapped by liposome. It was found that the VpGH could promote growth of the pigs.2...