Construction And Expression Analysis Of Recombination Plasmid Included HA2 And FimA Of Porphyromonas Gingivalis

Objective: Constructed stable and efficiented recombinant plasmids p VAX1-HA2-fim A/IL-15, p VAX1-HA2/IL-15, p VAX1-HA2-fim A, p VAX1-fim A/IL-15 and p VAX1-HA2 with the core functional areas HA2 of Porphyromonas gingivalis, the fimbriae protein gene fim A of P. gingivalis as target genes, and IL-15 gene of mice as the immune adjuvant. This article aimed to provide basic research and material conditions for next experiments.Methods: The article included three parts:Section I: Construction of the eukaryotic expression plasmids pVAX1-HA2-fim A/IL-15, p VAX1-HA2/IL-15, p VAX1-HA2-fim A, p VAX1-fim A/IL-15 and p VAX1-HA2.1. Total DNA extraction of P. gingivalis, total RNA extraction and reverse transcription of SD mice. Obtaining the target sequences of HA2, fim A, IL-15 and IRES, spliced those genes in series to get fragments HA2-fim A/IL-15, HA2/IL-15, HA2-fim A, fim A/IL-15 and HA2, meanwhile induced the enzyme cutting site Xho I and Nhe I.2. Digestion of the target fragments HA2-fim A/IL-15, HA2/IL-15, HA2- fim A, fim A/IL-15, HA2 and vector p VAX1 by double enzyme.3. Spliced the digested plasmid p VAX1 fragments with the target genes, constructed the recombinant plasmids p VAX1-HA2-fim A/IL-15, p VAX1-HA2/IL-15, p VAX1-HA2-fim A, p VAX1-fim A/IL-15 and p VAX1-HA2. All recombinant plasmids were transformed in DH5α cells. Positive clones were screened and identified by DNA sequencing method.Section 2: The expression of recombinant plasmids in eukaryotic cells.1. The cultivation of 293 T cells.2. All recombinant plasmids were transfected in 293 T cells, the blank control group was also constructed. Total RNA of transfected 293 T cells were extracted after 24 h, then conducted reverse transcription by stem rings method.3. Specific primers were designed respectively according to the target genes, PCR products were verified by agarose gel electrophoresis, corresponding sequencing results were tested by BLAST in NCBI.Section 3: the expression of IL-15 protein in eukaryotic cells.1. Cultivation and transfection of the 293 T cells.2. The expression of IL-15 were detected by ELISA.(1) Extraction of the total proteins and the filtrate proteins of 293 T cells.(2) Drawing the standard curve of IL-15 protein, testing the target proteins by ELISA, corresponding experimental results were analysised.Results:1. The recombinant plasmids p VAX1-HA2-fim A/IL-15, p VAX1-HA2/IL-15, p VAX1-HA2-fim A, p VAX1-fim A/IL-15 and p VAX1-HA2 were identified by enzyme digestion and sequencing.The target genes HA2, fim A and IL-15 were directional inserted into the eukaryotic expression plasmid p VAX1, the insert position was correct and the reading frames of the target genes had not been changed.2. All five recombinant plasmids were used to transfect 293 T cells. The target genes HA2, fim A and IL-15 were validated by RT-PCR and we got clear single target bands with correct size. The sequencing results were consistent with the published sequences in NCBI.3. IL-15 standard curve was drew. Spss analysis showed that, when compared the groups of p VAX1-HA2-fim A/IL-15, p VAX1-HA2/IL-15 and p VAX1-fim A/IL-15 with the negative/blank control group, the differences were statistically significant(p﹤0.05).Conclusion: Recombinant plasmids p VAX1-HA2-fim A/IL-15, p VAX1-HA2/IL-15, p VAX1-HA2-fim A, p VAX1-fim A/IL-15 and p VAX1-HA2 were construction successfully. In vitro RT-PCR was conducted to verify the expression of above m RNA correctly. ELISA were also used to verify the expression of IL-15 protein.,and the expression of IL-15 protein in eukaryotic cells after transfected by p VAX1-HA2-fim A/IL-15, p VAX1-HA2/IL-15 and p VAX1-fim A/IL-15 were also tested.