CAJ | 학술논문

目的在HBsAg DNA疫苗质粒中共表达IL-12佐剂分子,研究IL-12的表达水平对于HBV DNA疫苗质粒免疫原性的影响.方法构建携带来源于中国地区C型HBV参照序列CHN-HBV07-C经密码子优化的preS2-S基因的DNA疫苗质粒pHBV,并将3个不同IL-12表达水平的佐剂分子表达盒序列分别克隆入该疫苗质粒中,通过瞬时转染293T细胞以检测重组质粒IL-12分子及乙肝表面抗原的表达情况.将不同IL-12表达水平的疫苗质粒免疫BALB/c雌性小鼠,IFN-γ的ELISPOT方法检测HBsAg抗原特异性的细胞免疫应答,化学发光定量ELISA法检测HBsAg特异的抗体水平.结果成功构建3个不同IL-12表达水平的HBV DNA疫苗质粒,293T细胞转染结果显示:不携带IL-12分子表达盒的对照疫苗质粒pHBV的HBsAg表达水平可达70 ng/ml;低表达IL-12的疫苗质粒pHBV-12l的HBsAg表达水平为18 ng/ml;而高表达IL-12的重组疫苗质粒pHBV-12h的HBsAg表达水平仅为6 ng/ml.BALB/c小鼠的免疫结果表明:高表达IL-12分子的疫苗质粒pHBV-12h所诱导的细胞免疫及体液免疫水平相对于对照疫苗质粒pHBV均显著降低了.低表达IL-12分子的疫苗质粒pHBV-12l所诱导的抗体水平也显著降低了,但其细胞免疫应答水平却显著提高了.结论在同一疫苗质粒中,高表达IL-12分子的质粒可能会影响到抗原蛋白的表达,而低表达IL-12分子的疫苗质粒却能增强细胞免疫应答.因此,平衡好佐剂分子及抗原蛋白的表达水平对于诱导高水平的免疫应答是个重要的因素.
목적 재HBsAg DNA역묘질립중공표체IL-12좌제분자,연구IL-12적표체수평대우HBV DNA역묘질립면역원성적영향.방법 구건휴대래원우중국지구C형HBV삼조서렬CHN-HBV07-C경밀마자우화적preS2-S기인적DNA역묘질립pHBV,병장3개불동IL-12표체수평적좌제분자표체합서렬분별극륭입해역묘질립중,통과순시전염293T세포이검측중조질립IL-12분자급을간표면항원적표체정황.장불동IL-12표체수평적역묘질립면역BALB/c자성소서,IFN-γ적ELISPOT방법검측HBsAg항원특이성적세포면역응답,화학발광정량ELISA법검측HBsAg특이적항체수평.결과 성공구건3개불동IL-12표체수평적HBV DNA역묘질립,293T세포전염결과현시:불휴대IL-12분자표체합적대조역묘질립pHBV적HBsAg표체수평가체70 ng/ml;저표체IL-12적역묘질립pHBV-12l적HBsAg표체수평위18 ng/ml;이고표체IL-12적중조역묘질립pHBV-12h적HBsAg표체수평부위6 ng/ml.BALB/c소서적면역결과표명:고표체IL-12분자적역묘질립pHBV-12h소유도적세포면역급체액면역수평상대우대조역묘질립pHBV균현저강저료.저표체IL-12분자적역묘질립pHBV-12l소유도적항체수평야현저강저료,단기세포면역응답수평각현저제고료.결론 재동일역묘질립중,고표체IL-12분자적질립가능회영향도항원단백적표체,이저표체IL-12분자적역묘질립각능증강세포면역응답.인차,평형호좌제분자급항원단백적표체수평대우유도고수평적면역응답시개중요적인소.
Objective To investigate the effects of IL-12 coexpression level on antigen expression and immune responses induced by HBsAg DNA vaccination. Methods DNA vaccine plasmid pHBV carrying codon-optimized preS2-S gene of reference sequence CHN-HBV07-C in China was constructed. Three DNA vaccine plasmids pHBV-12i, pHBV-12l and pHBV-12h were also constructed by subcloning three different IL-12 expression cassettes with various expression strengths to plasmid pHBV, respectively. Expression levels of IL-12 and HBsAg in vaccine plasmid-tranfected 293T cells were measured by quantitative ELISA. DNA vaccines were administered intramuscularly to BALB/c mice and HBsAg-specific cellular immune responses were determined by IFN-γ ELISPOT. HBsAg-specific antibodies were tested by Chemiluminescence Quantitative Immunoassay. Results The HBsAg expression level in 293T cells was 70 ng/ml when transfected by plasmid pHBV without IL-12 expression cassette, and the HBsAg level was 18 ng/ml when transfected by plasmid pHBV-12l carrying low-level IL-12 expression cassette, whereas the HBsAg level was only 6 ng/ml when transfected by plasmid pHBV-12h carrying high-level IL-12 expression cassette.Results of DNA vaccination revealed that HBsAg-specific humoral and cellular immune responses were significantly decreased in mice administering vaccine pHBV-12h carrying high-level IL-12 expression cassette. Although HBsAg-specific antibody responses in mice inoculated with pHBV-12l were also decreased when compared with those in pHBV-vaccinated mice without IL-12 expression, the HBsAg-specific cellular immune responses were significantly increased. Conclusion High-level coexpression of IL-12 may suppress the expression of HBsAg, Whereas modest coexpression of IL-12 significantly enhanced the HBsAg-specific T cell responses induced by DNA vaccination. Therefore, it is so important to balance the expression between adjuvant and antigen to enhance the immune response.