| The repeated relapses obstructed the development of the therapy of the lymphoma. The main reason of the relapse is the existence of the residual tumor cells. The idiotype of membranous immunoglobulin of the B-cell lymphoma is subject to the ideal target of the DNA vaccine, an emerged potent strategy to treat and prevent the relapse of the tumor. The purpose of this study is to construct DNA vaccine against the idiotype of the clinical bioptic lymphoma tissue and a human B-cell lymphoma cell line, Namalwa, to evaluate the humourial and celluar immuno responses in vivo. The results whould provide the basis for further studies and optimization of this therapeutic strategy on patients with B-lymphoproliferative disease.Brifely, three idiotypic gene fragments including complementary-determining regions 3(CDR3), heavy chain variable region (VH) and single chain Fv (scFv) were amplified by means of RT-PCR. In order to enhance the immunigenicity of the fragments, murine monocyte chemotic protein-3(MCP-3) intact cDNA was cloned and fused with the 3 cloned idiotypic gene fragments respectively. The resulted 3 fusion sequences were subcloned into pcDNA3.1 to construct 3 DNA vaccine plasmids. Subsequently, the 4 groups of mice were immunized with 3 DNA vaccines and the mock plasmid cDNA3.1 respectivly to evaluated the specific anti-idiotypic immune responses in vivo.Firstly, we constructed 3 DNA vaccines containing CDR3, VH and scFv gene fragments respectively. The candidate gene fragment,VH of human B-cell lymphoma cell line Namalwa and VH, VL of the clinical bioptic tissue, were amplified using Ig superfamily primers by RT-PCR.The VH, VL framents of the clinical bioptic tissue were fused together with 15aa linker peptide to assemble scFV using classic method. Acording to the DNA alignment with the cloned Namalwa VH sequence against human Ig data deposited in Gene Bank, CDR3 sequence of VH was identified. Next, CDR3 and tnurine MCP-3 sequences were modified by PCR to facilitate fusion of the two sequences with 8aa linker peptide by recombinant PCR. Using the same strategy, we acquire MCP-3 fused Namalwa VH and biopcy scFV respectively. The 3 fusion gene fragments were then subcloned into eukaryonic expression vector pcDNA3.1 to construct DNA vaccine plamids.Secondly, the three constructed recombinant plasmids were extracted and purified in large scale. In advance to immunization, transient transfection coupled with RT-PCR proved that the recombinant plasmids could express in eukaryonic cells in right way. Then four groups of mice were immunized by intramuscular injection with different DNA vaccines and mock plasmid pcDNA3.1 respectively. The first group was vaccinated with the plasmid pcDNA3.1/MCP-CDR3; the second was vaccinated with the plasmid pcDNA3.1/MCP-NVH; the third was vaccinated with the plasmid pcDNA3.1/MCP-scFv; the fourth injected with the empty plasmid pcDNA3.1 was used as control. Three times injections were performed with 100ug plasmid respectively at the beginning of the experiment and 2, 4 weeks after the first injection for all the groups. FACS analysis was chosen to detect the antibodies recognizing lymphoma cells at different time following vaccination. The cellular immune responses, activation of CTL, were evaluated by LDHreleasing method.Anti-idiotypic antibodies were elicited in three of five mice of the CDR3 group and the VH group, and in four of five mice of the scFv group. It was demonstrated that specific anti-idiotypic antibodies titer increased significantly in all the group of DNA vaccine immunized mice as early as the eight weeks after the first immunization. The antibodies titer increased stepwisely during the whole detection period of 20 weeks. There is no antibody against Namalwa detected in the sera of mock pcDNA3.1 immunized mice by FACS analysis. Further test indicated that the anti-idiotypic antibodies could maintain for at least twenty weeks with high titer. The titer of the antibodies between pcDNA3.1/MCP-CDR3 and pcDNA3.1/MCP-NVH immunized groups had no remar...
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