ABSTRACT
<p><b>OBJECTIVE</b>To develop a novel dual-modified vaccine, the superantigen-linked intestine-carcinoma cells expressing membrane-bound heat shock protein 70 (HSP70), and further examine its anticancer therapeutic effect.</p><p><b>METHODS</b>The pre-established intestine carcinoma CT26 line expressing membrane-bound heat shock protein 70 (HSP70) was amplified and incubated with superantigen fusion protein, staphylococcal enterotoxin A (SEA) fused with transmembrane sequence (SEA-TM), thereby the dual-modified vaccine was prepared after inactivation. The anticancer efficacy of the vaccine was examined.</p><p><b>RESULTS</b>The laser confocal microscopy and flow cytometry showed that there co-existed much HSP70 and SEA on the vaccine membrane surface. Both of the single-modified vaccines, the SEA-linked vaccine and membrane-bound-HSP70-expressing one, displayed marked tumor suppression, a prolonged survival period, augmented lymphocyte proliferation and higher NK and CTL activity in the vaccinated mice when compared with its counterpart. Furthermore, the dually modified vaccine induced lymphocyte proliferation most intensively, generated the highest NK and CTL activity as well as the strongest tumor rejection in the vaccinated mice. The survival period of the mice was further prolonged.</p><p><b>CONCLUSION</b>A new vaccine, SEA-linked and membrane-bound-HSP70-expressing intestine-carcinoma cells can induce more potent anticancer immunity and produce better therapeutic efficacy.</p>
Subject(s)
Animals , Mice , Cancer Vaccines , Therapeutic Uses , Cell Line, Tumor , Cell Membrane , Metabolism , Enterotoxins , Allergy and Immunology , Gene Expression , Genetic Vectors , HSP70 Heat-Shock Proteins , Genetics , Allergy and Immunology , Mice, Inbred BALB C , Superantigens , Allergy and Immunology , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To construct a chimeric SEA-hPLAP-1 cDNA with gene splicing by overlap extension.</p><p><b>METHODS</b>The SEA gene and a DNA fragment encoding the signal for GPI-anchor attachment of hPLAP -1 were amplified by PCR. The two amplified gene sequence was annealed to form a chimeric GPI- anchored SEA molecule with gene splicing by overlap extension. The resulting chimera was cloned in pGEM-T vector and verified by sequencing analysis.</p><p><b>RESULT</b>A chimeric SEA-hPLAP-1 cDNA was successfully constructed with gene splicing by overlap extension.</p><p><b>CONCLUSION</b>Gene splicing by overlap extension is a successful specific PCR technique for gene recombination.</p>
Subject(s)
Alkaline Phosphatase , Base Sequence , Enterotoxins , Genetics , GPI-Linked Proteins , Isoenzymes , Genetics , Molecular Sequence Data , Polymerase Chain Reaction , RNA Splicing , Recombinant Fusion Proteins , GeneticsABSTRACT
OBJECTIVE: To clone the transmembrane (TM) domain sequence of EGFR gene and lay a good foundation for constructing the transmembrane expression vector of recombinant superantigens and cytokines. METHODS: A pair of primers special to the sequence encoding TM domain of EGFR gene were synthesized, TM domain fragment was cloned by RT-PCR, and the PCR product of TM domain sequence was ligated with the pGEM-T vector and confirmed by DNA sequencing. RESULTS: TM domain sequence was successfully cloned and verified by DNA sequencing. CONCLUSION: The successful cloning of TM domain sequence provides a basis for the construction of transmembrane fusion protein of Superantigen-TM or Cytokines-TM in cancer biotherapy.