ABSTRACT
<p><b>Objective</b>To construct a GFP-fused mouse Parkin co-regulated gene (PACRG) baculovirus recombinant PACRG/GFP-pFastBac1 vector and express the fusion protein in Sf9 insect cells.</p><p><b>METHODS</b>Full-length mouse PACRG cDNA was amplified by PCR and cloned in frame to the vector pFastBac1 with eGFP (rpFBac-PACRG-GFP recombinant vector). The plasmid was transformed into DH10Bac cells to obtain the recombinant bacmid plasmid, the bacmid was transfected into Sf9 insect cells, and the expressed PACRG/GFP fusion protein was analyzed by Western blot and fluorescence microscopy.</p><p><b>RESULTS</b>The construction of the PACRG/GFP-pFastBac1 baculovirus plasmid was confirmed by sequencing and restriction enzyme digestion. Western blot showed the expression of the fusion protein carrying a green fluorescence in the Sf9 insect cells.</p><p><b>CONCLUSIONS</b>Conclusion: A PACRG/GFP-pFastBac1 recombinant baculovirus vector was successfully constructed and the fusion protein was highly expressed in the Sf9 insect cells. Our findings have provided a basis for further studies on the structure of the PACRG protein and regulation of spermatogenesis.</p>
Subject(s)
Animals , Mice , Baculoviridae , Blotting, Western , DNA, Complementary , Genetic Vectors , Green Fluorescent Proteins , Plasmids , Polymerase Chain Reaction , Proteins , Genetics , Metabolism , Recombinant Fusion Proteins , Sf9 Cells , TransfectionABSTRACT
Objective To establish an expression system of TCRγ9/δ2-Fc protein by baculovirus vector ex-pression system and identify biological function of expressed TCRγ9/δ2-Fc protein. Methods γ9Fc and 82 (OT3) Fc gene fragments were amplified by overlap PCR and inserted into expression vector pBACp10ph. The recombinant plasmid pBACp10ph-γ9/δ2(OT3)-Fc and the baculovirus DNA were co-transfected into st9 cells. The expression position of TCRγ9/δ2 (OT3)-Fc was identified by Western blot and the expression efficiency of γ9Fc and δ2 (OT3) Fc was tested by flow cytometry (FCM). Furthermore, the binding activity of TCRγ9/δ2 (OT3)-Fc protein with SKOV3 ceils and MNS protein was evaluated with laser scanning confocal microslopy and surface plasmon resonance (SPR). Results The recombinant vector pBACp10ph-γ9/δ2(OT3)-Fc was constructed and TCRγ9/δ2(OT3)-Fc protein was expressed in sf9 ceils. However, the expression efficiency of γ9Fc and 82 (0T3) Fc was quite differ-ent. It was proved that purified TCRγ9/δ2 (OT3)-Fc protein can bind with SKOV3 cell and MNS protein. Conclu-sion TCRγ9/δ2-Fc protein is successfully expressed in baculovirus vector expression system and TCRγ9/δ2-Fc protein can simulate the binding activity of TCR in vitro.
ABSTRACT
Objective To establish an expression system of TCR?9/?2-Fc protein by baculovirus vector expression system and identify biological function of expressed TCR?9/?2-Fc protein.Methods ?9Fc and ?2(OT3)Fc gene fragments were amplified by overlap PCR and inserted into expression vector pBACp10ph.The recombinant plasmid pBACp10ph-?9/?2(OT3)-Fc and the baculovirus DNA were co-transfected into sf9 cells.The expression position of TCR?9/?2(OT3)-Fc was identified by Western blot and the expression efficiency of ?9Fc and ?2(OT3)Fc was tested by flow cytometry(FCM).Furthermore,the binding activity of TCR?9/?2(OT3)-Fc protein with SKOV3 cells and MNS protein was evaluated with laser scanning confocal microslopy and surface plasmon resonance(SPR).Results The recombinant vector pBACp10ph-?9/?2(OT3)-Fc was constructed and TCR?9/?2(OT3)-Fc protein was expressed in sf9 cells.However,the expression efficiency of ?9Fc and ?2(OT3)Fc was quite different.It was proved that purified TCR?9/?2(OT3)-Fc protein can bind with SKOV3 cell and MNS protein.Conclusion TCR?9/?2-Fc protein is successfully expressed in baculovirus vector expression system and TCR?9/?2-Fc protein can simulate the binding activity of TCR in vitro.
ABSTRACT
Bovine growth hormone (bGH) gene was expressed in an insect spodoptera frugiperda cell line using a Baculovirus, Hyphantria cunea nuclear polyhedrosis virus (HcNPV). The bGH gene in pbGH plasmid was sequenced and amplified by PCR technique with two primers containing NcoI sites. The bGH gene consisted of 654 bp (217 amino acid residues), the 5'-untranslated region of the cloned bGH cDNA contains 56 bp, and the 3'-untranslated region contains 145 bp and two pallindromic regions. The amplified bGH gene DNA fragment (654 bp) was inserted into the NcoI site of the pHcEVII vector, which was named pHcbGH. The pHcbGH transfer vector DNA and the wild type HcNPV DNA were cotransfected into s. frugiperda cells to construct a recombinant virus. Eight recombinant viruses were selected and named HcbGH. One clone, HcbGH-4-1 showed largest plaque size, therefore the recombinant virus was further studied. The multiplication patters of the recombinant HcbGH-4-1 was similar to that of the wild type HcNPV. The bGH gene DNA in the HcbGH-4-1 recombinant was confirmed by Southern lot hybridization. The amount of the bGH (217 amino acid residues, 21 kDa) produced in S. frugiperda cells infected with the HcbGH-4-1 recombinant was approximately 5.5 ng per ml (106 cells) by radioimmunoassay.