ABSTRACT
The aim was to construct a prokaryotic expression plasmid encoding the fusion gene of single-chain variable fragment and monocyte chemotactic protein-3 (MCP-3). The cDNAs of immunoglobulin (Ig) VH and Ig VL were amplified by RT-PCR and assembled into the single-chain variable fragment (scFv) by recombinant PCR method. The cDNAs of Ig VH and Ig VL were connected by a (Gly4Ser)3 linker. Then, the fragments of scFv and MCP-3 were connected with a NDAQAPKS spacer, using recombinant PCR method again. The results indicated that the fusion gene of scFv-MCP-3 were constructed correctly and cloned into the prokaryotic expression plasmid successfully identified by sequencing and restriction endonucleases examination. Finally, the fusion protein was expressed in E coli DH5alpha under induction by arabinose. And the fusion protein was 65 kD and account for 30% of the total protein of the bacteria. In conclusion, a prokaryotic plasmid, encoding the fusion gene of single-chain variable fragment with MCP-3 and expressing idiotype protein vaccination against B cell lymphoma, was constructed correctly.
Subject(s)
Animals , Humans , Mice , Amino Acid Sequence , Cancer Vaccines , Genetics , Allergy and Immunology , Chemokine CCL7 , Genetics , Allergy and Immunology , Genetic Vectors , Immunoglobulin Idiotypes , Allergy and Immunology , Immunoglobulin Variable Region , Genetics , Allergy and Immunology , Lymphoma, B-Cell , Allergy and Immunology , Mice, Inbred BALB C , Molecular Sequence Data , Plasmids , Genetics , Prokaryotic Cells , Metabolism , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , Vaccines, DNA , Genetics , Allergy and ImmunologyABSTRACT
<p><b>AIM</b>The recombinant human smooth muscle 22 alpha (SM22alpha) was expressed by using Pichia pastoris.</p><p><b>METHODS</b>Using pGEM3z-SM22alpha as the template, SM22alpha coding region was amplified by PCR, and was inserted the expression vector pPIC9. Then the recombinant plasmid pPIC9-SM22alpha was transfected into Pichia pastoris. The products induced by methanol were precipitated by ammonium sulfate, then CM-cellulose chromatography was performed for SM22alpha. Polyclonal antibody against SM22alpha was produced by immunizing a rabbit with purified recombinant SM22alpha.</p><p><b>RESULTS</b>The positive clone with SM22alpha got high output at 84 hours after induction by methanol. The SM22alpha prepared by ammonium sulfate fractionation and chromatographic separation showed a single band whose apparent molecular weight was 22 kD on SDS-PAGE. Polyclonal antibody against SM22alpha could detect the SM22alpha expression in human or rat vascular walls.</p><p><b>CONCLUSION</b>High-level expression of SM22alpha is successfully achieved in Pichia pastoris. Antibody against SM22alpha can be used to explore the function of SM22alpha.</p>