Construction of pcDNA3. 1-Brugia malayi glyceraldehyde phosphate dehydrogenase eukaryotic recombinant plasmid and study on its role in DNA immunity / 中华传染病杂志

ABSTRACT

Objective To construct the pcDNA3. 1-Brugia malayi (Bm) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) eukaryotic recombinant plasmid and to study its effect on mouse cellular immunity response. Methods Total RNA was prepared from periodic Bm. The target gene fragments were amplified by reverse transcription-polymerase chain reaction (RT-PCR) technique and then were inserted into the cloning vector. pGEM-T Easy, and sub-cloned into pcDNA3. 1. Purified pcDNA 3. 1-BmGAPDH recombinant plasmid and CpG were injected into the anterior tibial muscle of BALB/c mice in order to induce host immunity response. Mice injected with PBS and mice injected with blank plasmid were prepared as controls. The mouse models were immunized for 3 times with an interval of 2 weeks. RT-PCR was utilized to detect target gene expression in the muscle tissue. MTT method was used to measure the immunized mice T lymphocytes stimulation index, while enzyme-linked immunoassay (ELISA) was used to determine the serum interleukin (IL)-4 and interferon (IFN)-γ level. Means were compared using t test with SPSS software. Results The recombinant plasmid pcDNA3. 1-BmGAPDH was constructed sucessfully. The target gene was 1020 bp long and its homology with known gene sequence in database was 99%. BmGAPDH gene in the injected muscle of the immunized mice was detected by PCR. The proliferation of spleen T lymphocytes was higher in pcDNA3-BmGAPDH group than in the 2 control groups which were 1. 398, 1. 006 and 1. 017,respectively (P< 0. 05). The levels of IFN-γ and IL-4 in serums from the immunized mice were significantly higher than those of the PBS control group and blank plasmid control group which were 163.905, 58.589, 51. 317 and 107. 906, 27.111, 34.627, respectively (P<0. 05). Immune adjuvant CpG could accelerate and boost antigen-specific immune responses induced by vaccine, which presented as significant increase of IFN-γ and lymphocyte proliferation at 4 weeks after immunization.Conclusion The recombinant eukaryotic plasmid pcDNA3. 1-BmGAPDH is constructed and could elicit cellular immune responses in immunized mice.