The expression and preliminary evaluation of HPV6bL2△N360E7E6 fusion protein in E.coli for genital warts

Objective To express HPV6bL2△N360E7E6 fusion protein in E.coli and preliminarily evaluate its immune effect.Methods Three HPV6b gene fragments,which were L2(1-360 bp),E7 and E6,were fused by overlapping PCR,then were inserted into a prokaryotic expression vector and expressed in E.coli.C57BL/6 mice were immunized with purified fusion protein plus Al(OH)3 and/or CpG adjuvants through intramuscular route,the cellular and humoral immune responses were detected by IFN-γ ELISPOT and ELISA respectively.Results Protein plus CpG adjuvant could induce the strongest cellular immune response to E7 and E6,high antibody titer against L2 could be detected in all immunized groups but there were no significant difference among these groups.Conclutions HPV6bL2△N360E7E6 gene was successfully cloned into pQE30 vector and expressed in E.coli,the fusion protein was also purified and proved that could induce strong cellular and humoral immune responses with appropriate adjuvant in C57 BL/ 6 mice and could be used for future research.     

人乳头瘤病毒11型L1/E7嵌合DNA疫苗的构建及其诱导的免疫效应

Objective To construct chimerical DNA vaccine plasmid of human papiUomavirus type 11 (HPV11) L1-E7, and to evaluate its immunogenicity. Methods Molecular cloning techniques were used to construct recombinant plasmid PeDNA3 L1-E7 as a DNA vaccine. BALB/c mice were vaccinated with DNA recombinants through muscle injection. IL-2 and γ-INF secreted by immunized spleens lymphocyte and HPV 11 LI or E7 specific antibodies were assayed by ELISA method. Spleens lymphocyte proliferation was measured by MTT assay. Results The chimerical DNA plasmid of pcDNA3 LI-E7 was constructed correctly. Specific anti-HPV11 E7 and L1 antibodies, specific lymphocyte proliferation and secretions of IL-2 and γ-INF were detected in vaccinated mice. Conclusion Specific immune response, including cellular immunity and humoral immunity, could been detected in mice vaccinated with chimerical DNA vaccine of pcDNA3 L1-E7.     

人乳头瘤病毒11型L1/E7嵌合DNA疫苗的构建及其诱导的免疫效应

Objective To construct chimerical DNA vaccine plasmid of human papiUomavirus type 11 (HPV11) L1-E7, and to evaluate its immunogenicity. Methods Molecular cloning techniques were used to construct recombinant plasmid PeDNA3 L1-E7 as a DNA vaccine. BALB/c mice were vaccinated with DNA recombinants through muscle injection. IL-2 and γ-INF secreted by immunized spleens lymphocyte and HPV 11 LI or E7 specific antibodies were assayed by ELISA method. Spleens lymphocyte proliferation was measured by MTT assay. Results The chimerical DNA plasmid of pcDNA3 LI-E7 was constructed correctly. Specific anti-HPV11 E7 and L1 antibodies, specific lymphocyte proliferation and secretions of IL-2 and γ-INF were detected in vaccinated mice. Conclusion Specific immune response, including cellular immunity and humoral immunity, could been detected in mice vaccinated with chimerical DNA vaccine of pcDNA3 L1-E7.     

人乳头瘤病毒11型L1/E7嵌合DNA疫苗的构建及其诱导的免疫效应

Objective To construct chimerical DNA vaccine plasmid of human papiUomavirus type 11 (HPV11) L1-E7, and to evaluate its immunogenicity. Methods Molecular cloning techniques were used to construct recombinant plasmid PeDNA3 L1-E7 as a DNA vaccine. BALB/c mice were vaccinated with DNA recombinants through muscle injection. IL-2 and γ-INF secreted by immunized spleens lymphocyte and HPV 11 LI or E7 specific antibodies were assayed by ELISA method. Spleens lymphocyte proliferation was measured by MTT assay. Results The chimerical DNA plasmid of pcDNA3 LI-E7 was constructed correctly. Specific anti-HPV11 E7 and L1 antibodies, specific lymphocyte proliferation and secretions of IL-2 and γ-INF were detected in vaccinated mice. Conclusion Specific immune response, including cellular immunity and humoral immunity, could been detected in mice vaccinated with chimerical DNA vaccine of pcDNA3 L1-E7.     

人乳头瘤病毒11型L1/E7嵌合DNA疫苗的构建及其诱导的免疫效应

Objective To construct chimerical DNA vaccine plasmid of human papiUomavirus type 11 (HPV11) L1-E7, and to evaluate its immunogenicity. Methods Molecular cloning techniques were used to construct recombinant plasmid PeDNA3 L1-E7 as a DNA vaccine. BALB/c mice were vaccinated with DNA recombinants through muscle injection. IL-2 and γ-INF secreted by immunized spleens lymphocyte and HPV 11 LI or E7 specific antibodies were assayed by ELISA method. Spleens lymphocyte proliferation was measured by MTT assay. Results The chimerical DNA plasmid of pcDNA3 LI-E7 was constructed correctly. Specific anti-HPV11 E7 and L1 antibodies, specific lymphocyte proliferation and secretions of IL-2 and γ-INF were detected in vaccinated mice. Conclusion Specific immune response, including cellular immunity and humoral immunity, could been detected in mice vaccinated with chimerical DNA vaccine of pcDNA3 L1-E7.     

人乳头瘤病毒11型L1/E7嵌合DNA疫苗的构建及其诱导的免疫效应

Objective To construct chimerical DNA vaccine plasmid of human papiUomavirus type 11 (HPV11) L1-E7, and to evaluate its immunogenicity. Methods Molecular cloning techniques were used to construct recombinant plasmid PeDNA3 L1-E7 as a DNA vaccine. BALB/c mice were vaccinated with DNA recombinants through muscle injection. IL-2 and γ-INF secreted by immunized spleens lymphocyte and HPV 11 LI or E7 specific antibodies were assayed by ELISA method. Spleens lymphocyte proliferation was measured by MTT assay. Results The chimerical DNA plasmid of pcDNA3 LI-E7 was constructed correctly. Specific anti-HPV11 E7 and L1 antibodies, specific lymphocyte proliferation and secretions of IL-2 and γ-INF were detected in vaccinated mice. Conclusion Specific immune response, including cellular immunity and humoral immunity, could been detected in mice vaccinated with chimerical DNA vaccine of pcDNA3 L1-E7.     

人乳头瘤病毒11型L1/E7嵌合DNA疫苗的构建及其诱导的免疫效应

Objective To construct chimerical DNA vaccine plasmid of human papiUomavirus type 11 (HPV11) L1-E7, and to evaluate its immunogenicity. Methods Molecular cloning techniques were used to construct recombinant plasmid PeDNA3 L1-E7 as a DNA vaccine. BALB/c mice were vaccinated with DNA recombinants through muscle injection. IL-2 and γ-INF secreted by immunized spleens lymphocyte and HPV 11 LI or E7 specific antibodies were assayed by ELISA method. Spleens lymphocyte proliferation was measured by MTT assay. Results The chimerical DNA plasmid of pcDNA3 LI-E7 was constructed correctly. Specific anti-HPV11 E7 and L1 antibodies, specific lymphocyte proliferation and secretions of IL-2 and γ-INF were detected in vaccinated mice. Conclusion Specific immune response, including cellular immunity and humoral immunity, could been detected in mice vaccinated with chimerical DNA vaccine of pcDNA3 L1-E7.     

人乳头瘤病毒11型L1/E7嵌合DNA疫苗的构建及其诱导的免疫效应

Objective To construct chimerical DNA vaccine plasmid of human papiUomavirus type 11 (HPV11) L1-E7, and to evaluate its immunogenicity. Methods Molecular cloning techniques were used to construct recombinant plasmid PeDNA3 L1-E7 as a DNA vaccine. BALB/c mice were vaccinated with DNA recombinants through muscle injection. IL-2 and γ-INF secreted by immunized spleens lymphocyte and HPV 11 LI or E7 specific antibodies were assayed by ELISA method. Spleens lymphocyte proliferation was measured by MTT assay. Results The chimerical DNA plasmid of pcDNA3 LI-E7 was constructed correctly. Specific anti-HPV11 E7 and L1 antibodies, specific lymphocyte proliferation and secretions of IL-2 and γ-INF were detected in vaccinated mice. Conclusion Specific immune response, including cellular immunity and humoral immunity, could been detected in mice vaccinated with chimerical DNA vaccine of pcDNA3 L1-E7.     

人乳头瘤病毒11型L1/E7嵌合DNA疫苗的构建及其诱导的免疫效应

Objective To construct chimerical DNA vaccine plasmid of human papiUomavirus type 11 (HPV11) L1-E7, and to evaluate its immunogenicity. Methods Molecular cloning techniques were used to construct recombinant plasmid PeDNA3 L1-E7 as a DNA vaccine. BALB/c mice were vaccinated with DNA recombinants through muscle injection. IL-2 and γ-INF secreted by immunized spleens lymphocyte and HPV 11 LI or E7 specific antibodies were assayed by ELISA method. Spleens lymphocyte proliferation was measured by MTT assay. Results The chimerical DNA plasmid of pcDNA3 LI-E7 was constructed correctly. Specific anti-HPV11 E7 and L1 antibodies, specific lymphocyte proliferation and secretions of IL-2 and γ-INF were detected in vaccinated mice. Conclusion Specific immune response, including cellular immunity and humoral immunity, could been detected in mice vaccinated with chimerical DNA vaccine of pcDNA3 L1-E7.     

人乳头瘤病毒11型L1/E7嵌合DNA疫苗的构建及其诱导的免疫效应

Objective To construct chimerical DNA vaccine plasmid of human papiUomavirus type 11 (HPV11) L1-E7, and to evaluate its immunogenicity. Methods Molecular cloning techniques were used to construct recombinant plasmid PeDNA3 L1-E7 as a DNA vaccine. BALB/c mice were vaccinated with DNA recombinants through muscle injection. IL-2 and γ-INF secreted by immunized spleens lymphocyte and HPV 11 LI or E7 specific antibodies were assayed by ELISA method. Spleens lymphocyte proliferation was measured by MTT assay. Results The chimerical DNA plasmid of pcDNA3 LI-E7 was constructed correctly. Specific anti-HPV11 E7 and L1 antibodies, specific lymphocyte proliferation and secretions of IL-2 and γ-INF were detected in vaccinated mice. Conclusion Specific immune response, including cellular immunity and humoral immunity, could been detected in mice vaccinated with chimerical DNA vaccine of pcDNA3 L1-E7.     

人乳头瘤病毒11型L1/E7嵌合DNA疫苗的构建及其诱导的免疫效应

Objective To construct chimerical DNA vaccine plasmid of human papiUomavirus type 11 (HPV11) L1-E7, and to evaluate its immunogenicity. Methods Molecular cloning techniques were used to construct recombinant plasmid PeDNA3 L1-E7 as a DNA vaccine. BALB/c mice were vaccinated with DNA recombinants through muscle injection. IL-2 and γ-INF secreted by immunized spleens lymphocyte and HPV 11 LI or E7 specific antibodies were assayed by ELISA method. Spleens lymphocyte proliferation was measured by MTT assay. Results The chimerical DNA plasmid of pcDNA3 LI-E7 was constructed correctly. Specific anti-HPV11 E7 and L1 antibodies, specific lymphocyte proliferation and secretions of IL-2 and γ-INF were detected in vaccinated mice. Conclusion Specific immune response, including cellular immunity and humoral immunity, could been detected in mice vaccinated with chimerical DNA vaccine of pcDNA3 L1-E7.     

Immunogenicity and safety of liposome-vaccine encapsulating hepatitis B surface antigen and phosphodiester CpG oligodeoxynucleotides

CpG oligodeoxynucleotides (CpG ODN) as adjuvant have been extensively studied in recent years. Phosphodiester CpG ODN (PO CpG ODN) can perfectly mimic bacterial DNA in enhancing immune response but are vulnerable to nucleases in vivo . This study aimed to evaluate the immunostimulatory potential and safety of phosphodiester CpG ODN encapsulated in nonphospholipid liposomes. BALB/c mice were immunized intramuscularly with different formulations of liposomes,CpG ODN and hepatitis B surface antigen (HBsAg). The results demonstrated that the encapsulated PO CpG ODN were protected against rapid degradation in vivo and retained their adjuvant activity. PO CpG ODN encapsulated with HBsAg in liposomes induced strong Th1-biased or Th1/Th2 mixed humoral immune response in mice with the magnitude similar to their phosphothioate equivalent in the same formulation. High IFN-gamma production induced by this formulation confirmed the generation of strong cellular immune response. Additionally, co-delivery of HBsAg and PO CpG ODN improved the immune response over that obtained with separate delivery. Safety experiment showed that liposome-encapsulaed PO CpG ODN and HBsAg caused mild systemic and moderate local adverse reaction. In conclusion, our data shows that PO CpG ODN encapsulated in liposomes fully exhibit their Th1-type adjuvant activity and act as a potential adjuvant for vaccines.     

二甲基三十六烷基铵及卡介苗多糖核酸佐剂在结核亚单位疫苗加强BCG免疫中的效应研究

Objective To investigate the adjuvant effect of dimo-thylidioctyl ammonium bromide (DDA) and/or DDA-BCG polysaccharide nucleic acid( BCG-PSN), which was combined with a Mycobacterium tuberculosis fusion protein AMM ( Ag 8 5 B - MPT64190-198 - Mtb8.4 ) to boost BCG primed immunization. Methods DDA with or without BCG PSN was mixed with the fusion protein AMM to construct the boosting vaccine. Mice were immunized with BCG and then boosted twice with AMM formulated with the adjuvant DDA with or without BCG-PSN. PBS or BCG vaccination without boosting was used as control. The humoral and cell-mediated immune responses were analyzed by ELISA and ELISPOT. Moreover, the protective efficacy of BCG prime-AMM subunit vaccine boosting against Mycobacterium tuberculosis infection was analyzed. Results With in vitro stimulation of Ag85B and PPD( purified protein derivative) antigen, the number of IFN-γ secreting cells from the mice boosted twice by AMM/DDA/BCG-PSN and AMM/DDA were higher than BCG and PBS group (P <0.05). The CFU in lungs of mice boosted with AMM/DDA/BCG-PSN was less than that of PBS group(P <0.05), while the CFU of AMM/DDA-boosted mice was less than that of BCG and PBS group(P < 0.05).However, fewer lesions were seen in lungs of mice immunized with BCG alone or BCG-prime-AMM/DDA/BCG-PSN boosting than the other groups. Conclusion DDA is an idea adjuvant for tuberculosis subunit vaccine;BCG-PSN might play a role in alleviating the immunity-mediated pathology.     

二甲基三十六烷基铵及卡介苗多糖核酸佐剂在结核亚单位疫苗加强BCG免疫中的效应研究

Objective To investigate the adjuvant effect of dimo-thylidioctyl ammonium bromide (DDA) and/or DDA-BCG polysaccharide nucleic acid( BCG-PSN), which was combined with a Mycobacterium tuberculosis fusion protein AMM ( Ag 8 5 B - MPT64190-198 - Mtb8.4 ) to boost BCG primed immunization. Methods DDA with or without BCG PSN was mixed with the fusion protein AMM to construct the boosting vaccine. Mice were immunized with BCG and then boosted twice with AMM formulated with the adjuvant DDA with or without BCG-PSN. PBS or BCG vaccination without boosting was used as control. The humoral and cell-mediated immune responses were analyzed by ELISA and ELISPOT. Moreover, the protective efficacy of BCG prime-AMM subunit vaccine boosting against Mycobacterium tuberculosis infection was analyzed. Results With in vitro stimulation of Ag85B and PPD( purified protein derivative) antigen, the number of IFN-γ secreting cells from the mice boosted twice by AMM/DDA/BCG-PSN and AMM/DDA were higher than BCG and PBS group (P <0.05). The CFU in lungs of mice boosted with AMM/DDA/BCG-PSN was less than that of PBS group(P <0.05), while the CFU of AMM/DDA-boosted mice was less than that of BCG and PBS group(P < 0.05).However, fewer lesions were seen in lungs of mice immunized with BCG alone or BCG-prime-AMM/DDA/BCG-PSN boosting than the other groups. Conclusion DDA is an idea adjuvant for tuberculosis subunit vaccine;BCG-PSN might play a role in alleviating the immunity-mediated pathology.     

Expression,purification and characterization of Mycobacterium tuberculosis RpfE protein

Resuscitation promoting factor E(RpfE)is one of the five Rpf-like proteins in Mycobacterium tuberculosis(M.tuberculosis).These Rpf-like proteins are secretory,which make them candidates for recognition by the host immune system.In this study,the RpfE gene was amplified from M.tuberculosis,cloned into the expression vectors pDE22 and pPRO EXHT,and were expressed in Mycobacterium vaccae(M.vaccae)and Escherichia coli DH5α,respectively.Both recombinant RpfE proteins were purified by Ni-Sepharose affinity chromatography,and were given to C57BL/6 mice.The RpfE proteins elicited T cell proliferation,and stimulated the production of gamma interferon(IFN-γ),interleukin-10(IL-10)and IL-12.Our results indicated that the RpfE protein expressed in M.vaccae could more efficiently stimulate cellular immune response,making it a promising candidate as a subunit vaccine.     

人巨细胞病毒gB和pp150融合基因真核表达载体的构建及其免疫活性研究

Objective To provide experimental evidence for development of human cytomegalovirus (HCMV) nucleic acid vaccine,HCMV surface protein(gB),membrane protein(pplSO),and gB-pp150 fused gene eukaryotie expression vector were constructed.Methods gB and pp150 genes were amplified and fused into gB-pp150,then were cloned into pcDNA 3.1(+) to obtain recombinant expression plasmids pcDNA 3.1(+)-gB,pcDNA 3.1(+)-pp150 and pcDNA 3.1(+)-gB-pp150,which were encapsulated with chitosan.Mouse were vaccinated and the humoral and eell immune response were determined by ELISA,specific proliferative response of plenie lymphocytes.Results The gB,pp150 and gB-pp150 fusion gene eukaryotie expression vector were successfully constructed.The antibodies A value induced by peDNA3.1(+)-gB or peDNA3.1(+)-gB-pp150 were much higher than that of peDNA3.1(+)(P<0.01).The IFN-γ levels induced by pcDNA3.1(+)-pp150 and peDNA3.1(+)-gB-pp150 were significantly higher than that of pcDNA3.1(+).There are significant diferenee between the stimulating indexes of pcDNA3.1(+)-pp150 or peDNA3.1(+)-gB-pp150 immunized and normal mice.Conclusion The DNA vaccine pcDNA3.1(+)-gB can induce significant humeral immunity response.and pcDNA3.1 (+)-pp150 can induce high cellular immune response,whereas pcDNA3.1(+)-gB-pp150 can induce both humeral and cellar immune responses in BALB/c mice.     

人巨细胞病毒gB和pp150融合基因真核表达载体的构建及其免疫活性研究

Objective To provide experimental evidence for development of human cytomegalovirus (HCMV) nucleic acid vaccine,HCMV surface protein(gB),membrane protein(pplSO),and gB-pp150 fused gene eukaryotie expression vector were constructed.Methods gB and pp150 genes were amplified and fused into gB-pp150,then were cloned into pcDNA 3.1(+) to obtain recombinant expression plasmids pcDNA 3.1(+)-gB,pcDNA 3.1(+)-pp150 and pcDNA 3.1(+)-gB-pp150,which were encapsulated with chitosan.Mouse were vaccinated and the humoral and eell immune response were determined by ELISA,specific proliferative response of plenie lymphocytes.Results The gB,pp150 and gB-pp150 fusion gene eukaryotie expression vector were successfully constructed.The antibodies A value induced by peDNA3.1(+)-gB or peDNA3.1(+)-gB-pp150 were much higher than that of peDNA3.1(+)(P<0.01).The IFN-γ levels induced by pcDNA3.1(+)-pp150 and peDNA3.1(+)-gB-pp150 were significantly higher than that of pcDNA3.1(+).There are significant diferenee between the stimulating indexes of pcDNA3.1(+)-pp150 or peDNA3.1(+)-gB-pp150 immunized and normal mice.Conclusion The DNA vaccine pcDNA3.1(+)-gB can induce significant humeral immunity response.and pcDNA3.1 (+)-pp150 can induce high cellular immune response,whereas pcDNA3.1(+)-gB-pp150 can induce both humeral and cellar immune responses in BALB/c mice.     

Immunogenicity of the recombinant adenovirus type 5 vector with type 35 fiber containing HIV-1 gag gene

The immune efficiency of a recombinant adenovirus type 5 with type 35 fiber containing HIV-1 gag gene (rAd5/F35-mod.gag) was investigated in BALB/c mice, in which the rAd5/F35-mod.gag was firstly identified with PCR, then transfected to 293 cells and the in vitro expression level of Gag protein was determined by Western blotting and indirect immuno-fluorescent assay. Mice were immunized with intramuscular injections of rAd5/F35-mod.gag, rAd5-mod.gag or DNA and were boosted after 3 weeks. To test the effect of pre-existing anti-viral immunity on immunization, mice were also injected with Ad5-GFP vector and then immunized 4 and 7 weeks later with Ad5/F35-mod. gag vector. The P24-specific IgG antibody in sera of immunized mice was determined by ELISA and the specific cytotoxic T lymphocyte (CTL) response was assayed by intracellular cytokine staining. It was demonstrated that the rAd5/F35-mod. gag vector could express efficiently the HIV Gag protein in 293 cells in vitro and induce strong HIV-specific immune responses in vivo. The strongest CTL and serum IgG response occurred when mice were immunized twice with injection of rAd5/F35 alone, but the anti-Ad5 antibody after primary infection with adenovirus could inhibit the specific immune responses induced by rAd5/F35 vector. It is concluded that single immunization with recombinant adenovirus rAd5/F35-mod. gag can induce specific CTL and serum IgG antibody responses in mice, but the immunogenicity of rAd5/F35 is comparably weaker than that of rAd5.     

体内电穿孔在质粒介导的基因转移及DNA免疫中的应用

Objective To investigate the effects of in vivo electroporation on plasmid mediated reporter gene expression and immunogenicity of DNA vaccine. Methods Luciferase expression plasmid was administered intramuscularly to BALB/c mice at 8μg and 40μg dosage level through injection with or without eletroporation Luciferase expression level in murine muscle was detected by IVIS imaging system 24 h after injection. DNA vaccine plasmid p1.0-gp1455m carrying codon-optimized env gene of CN54 strain ( HIV-1 CRF07_BC) was administered to mice at dosages of 8μg and 40μg through the two approaches mentioned above. Mice were immunized at week 0,2 and 4. Env-specific immune responses were detected at two weeks post the second and the third vaccinations. Env-specific antibody immune responses were determined by ELISA. Euv-specific cellular immune responses were determined by IFN-γ ELISPOT. Results Luciferase expression level in murine muscle was significantly increased as much as 35 folds through in vivo eletroporation. Results of ELISA and ELISPOT revealed that in vivo eletroporation could significantly enhance both the humoral and cellular immune responses induced by DNA vaccination. The responses induced by electrodelivered p1.0-gp1455m at 8 μg dosage were better than those induced by simple intramuscular injection with 40 μg of plasmid DNA. On the other hand, 2 injections followed by electroporation elicited comparable level of humoral and cellular immune responses with those induced by 3 injections without electroporation. Conclusion In vivo electroporation was capable of enhancing both the plasmid-mediated gene expression and immunogenicity of DNA vaccine.     

体内电穿孔在质粒介导的基因转移及DNA免疫中的应用

Objective To investigate the effects of in vivo electroporation on plasmid mediated reporter gene expression and immunogenicity of DNA vaccine. Methods Luciferase expression plasmid was administered intramuscularly to BALB/c mice at 8μg and 40μg dosage level through injection with or without eletroporation Luciferase expression level in murine muscle was detected by IVIS imaging system 24 h after injection. DNA vaccine plasmid p1.0-gp1455m carrying codon-optimized env gene of CN54 strain ( HIV-1 CRF07_BC) was administered to mice at dosages of 8μg and 40μg through the two approaches mentioned above. Mice were immunized at week 0,2 and 4. Env-specific immune responses were detected at two weeks post the second and the third vaccinations. Env-specific antibody immune responses were determined by ELISA. Euv-specific cellular immune responses were determined by IFN-γ ELISPOT. Results Luciferase expression level in murine muscle was significantly increased as much as 35 folds through in vivo eletroporation. Results of ELISA and ELISPOT revealed that in vivo eletroporation could significantly enhance both the humoral and cellular immune responses induced by DNA vaccination. The responses induced by electrodelivered p1.0-gp1455m at 8 μg dosage were better than those induced by simple intramuscular injection with 40 μg of plasmid DNA. On the other hand, 2 injections followed by electroporation elicited comparable level of humoral and cellular immune responses with those induced by 3 injections without electroporation. Conclusion In vivo electroporation was capable of enhancing both the plasmid-mediated gene expression and immunogenicity of DNA vaccine.