Construction Of Human Cytomegalovirus IE1 DNA Vaccine And Primary Study Of Its Immunocompetence In Mice

Objective: To construct DNA vaccine containing HCMV IE1 and observe the humoral or celluar immune responses in BALB/c mice induced by the HCMV IE1 DNA vaccine injected by intramuscular cells, and to provide experimental basis for further studying DNA vaccine against HCMV infection.Methods:(1) IE1 fragment of the recombinant plasmid pEGFP-C1-IE1 cut by enzyme digestion was inserted into eukaryotic expression plasmid pcDNA3.1(-) and confirmed by EcoRⅠ/ HindⅢ.(2) The recombinant eukaryotic expression plasmid pcDNA3.1(-)-IE1 was transfected into HeLa cells. HCMV IE1 gene or protein was detected by RT-PCR or Western blot.(3) Eight weeks old BABL/c mice were immunized with pcDNA3.1(-)-IE1 or pcDNA3.1(-) or PBS buffer by intramuscular cells at 2-week interval for four times.(4) Two weeks after the last time of immunization, HCMV IE1 gene or protein in intramuscular cells was detected by PCR or observed with immunohistochemistry.(5) Two weeks after the last time of immunization, ELISA was used to detect the quantities of IL-4, IL-2 or IFN-γin the supernatant of murine spleen lymphocyte culture after stimulated by PHA.(6) The proliferation response of spleen cells was detect??sing MTT assay, and the stimulation indexs(SI) were counted. All of data were analyzed by SPSS13.0.Results:(1) The fragment of about 1476 bp, consistenting to HCMV IE1 gene, could be obtained from pcDNA3.1(-)-IE1 cut with EcoRⅠand HindⅢ.(2) The fragment of about 1476 bp, consistenting to HCMV IE1 gene, cound be amplified by RT-PCR. Meanwhile, HCMV IE1 protein with molecular weight about 72 kDa was detected using Western-blot in HeLa cells transfected with pcDNA3.1(-)-IE1.(3) In the intramuscular cells of immunized mice, about 1476 bp fragment, consistenting to HCMV IE1 gene was detected by PCR. HCMV IE1 gene cound exist constantly in intramuscular cells of mice. The result of immunohisto- chemistry showed that HCMV IE1 gene could express in intramuscular cells of mice.(4) IL-4 in mice inoculated with pcDNA3.1(-)-IE1 was 30.47±1.76 pg/mL, it was significant difference between the experiment group and the pcDNA3.1(-) group (26.88±2.60 pg/mL) or the PBS group (25.06±2.21 pg/mL) (P < 0.05); IL-2 in mice inoculated with pcDNA3.1(-)-IE1 was 47.98±2.08 pg/mL, it was significant difference between the experiment group and the pcDNA3.1(-) group (19.66±2.45 pg/mL)or the PBS group (16.06±2.94 pg/mL) (P < 0.001); IFN-γin mice inoculated with pcDNA3.1(-)-IE1 was 50.47±4.81 pg/mL, it was significant difference between the experiment group and the pcDNA3.1(-) group (28.24±4.18 pg/mL) or the PBS group (25.06±4.35 pg /mL)(P < 0.001).(5) The proliferation response of spleen cells of DNA vaccine group (SI:3.55±0.48) was significantly higher than those of mice injected with pcDNA3.1(-)(SI:1.86±0.23)or PBS (1.48±0.11) ( P < 0.001). Conclusion:(1) The eukaryotic expression recombinant pcDNA3.1(-)-IE1 was successfully constructed, HCMV IE1 gene could express in HeLa cells.(2) HCMV IE1 gene could exist and express in intramuscular cells of BALB/c mice.(3) The level of IL-4, IL-2 or IFN-r and stimulation indexs of spleen cells could be increased in BALB/c mice immunized with pcDNA3.1(-)-IE1 .