The Studies Of Construction、Expression And Immune Effects Of The Recombinant Plasmid Carrying HPV16E7c And HBD2Genes

Objectives:To construct the stably eukaryotic expression plasmid of pEGFP-Cl/HBD2-E7c fusion genes, and immunize the BALB/c mice with the plasmid and observe its immune function include the cell-mediated immune response.Methods:1. The gene of E7c was amplified by reverse polymerase chain reaction (PCR) from verified pcDNA3.1(+)/E7plasmid which can encode HPV16E7protein. The fragment by PCR was inserted into pcDNA3.1(+) to construct pcDNA3.1(+)/E7c, and Tightness of the gene sequence was identified by DNA sequencing.2. After the constructing commission of pEGFP-C1/HBD2was over by the microbiology company, the pcDNA3.1(+)/E7c and the pEGFP-C1/HBD2were digested by Apa I and EcoR I. Both digested products were ligated by T4DNA ligase to construct the plasmid pEGFP-C1/HBD2-E7c, and then transformed it into JM109with the product of the DNA linking reaction. The correctness of the positive clones were identified by Hind III and Xba I digestion and DNA sequencing. Then the recombinant plasmid carrying pEGFP-C1/HBD2-E7c genes was transfected into MCF-7cell by using lipofectin and the expression of pEGFP-C1/HBD2-E7c was examined by Immunofluorescence.3. Forty BALB/c mice were evenly divided into four groups randomly(10each), which was immunized with PBS100ul per time(A group), pcDNA3.1(+)/E7c100ug per time (B group), pEGFP-C1(+) and pcDNA3.1(+)/E7c(C group), pEGFP-Cl/HBD2-E7c (D group) through intramuscular injection respectively. The plasmid immunization scheme for each group was100ug/time and3times for each mouse totally, with14days equal interval during2immunizations.4. At the14th day after the last times of immunization, we dissected the mice, collected the serum of all mice to detect specific antibodies of E7by indirect ELISA. At the same time, the weights of the contact mouse body and the spleen isolated from them were weighted on electronic balance one by one, respectively, and the spleen index for each mouse was also calculated according to the formula. Then lymphocytes separated with the lymphocyte separation medium from splenocytes were stimulated by HPV16E7synthetic peptides, ConA or PBS to test their abilities of proliferation according to plan. At the same time, the counts of CD4positive T cells and CD8positive T cells and the CD4/CD8ratios of the spleen lymphocytes from each group were tested by FCM with fluorescent labeled specific antibodies of CD3, CD4and CD8.Results:1. The DNA sequencing result showed that the plasmid pcDNA3.1(+)/E7c and pEGFP-C1/HBD2-E7c was right. The successful constructions of eukaryotic expressing plasmids and multigene expressing plasmids were confirmed by restriction endonuclease digestion and DNA sequencing.2. The HPV16E7serum specific antibody was significant difference among4different groups of BALB/c mice immunized by different plasmids or PBS, which was test by analysis of variance (P<0.05). The group of pEGFP-C1/HBD2-E7c produced more HPV16E7specific antibody than others, which was verified by SNK statistical analysis (P<0.05).3. Compared with the spleen index of each group, there was significant difference among them (P<0.05), and the spleen index of pEGFP-C1/HBD2-E7c group was the lowest one(P<0.05), while the PBS group was the highest(P<0.05).4. According to the test of FCM which the molecules of CD3,CD4and CD8were labeled by the Percp, FITC and PE, respectively, the levels of CD3+,CD4+cells and CD3+,CD8+cells in pEGFP-C1/HBD2-E7c group were both the highest than other groups statistically (P<0.05). The percentages of the T helper lymphocytes (CD3+, CD4+) and cytotoxic T lymphocytes(CD3+,CD8+) of pEGFP-C1/HBD2-E7c group accounted42.52±4.42%and50.16±4%of all spleen lymphocytes for each, and the absolute count of these two kinds of cells reached4190±144and4970±140in average10000spleen lymphocytes; But CD4/CD8ratio of pEGFP-Cl/HBD2-E7c group was only0.84±0.03, which was the lowest one.Conclusions:1. The construction of eukaryotic expression plasmid of pEGFP-C1/HBD2- E7c was successful and its expression in MCF-7cells laid the foundation for the next step of the elevation of its immunological effects as DNA vaccine.2. The results show that as an adjuvant, human-beta defensin2could obviously enhanced the both effects of the specific humoral immunity and the cellular immunity of HPV16E7c gene, especially the CD8+T lymphocytes mediated cellular immunity.