中华预防医学杂志
中華預防醫學雜誌
중화예방의학잡지
CHINESE JOURNAL OF
2012年
1期
58-63
,共6页
吴娟%马辉%范小勇%曲勍%罗玉萍%Douglas B.Lowrie
吳娟%馬輝%範小勇%麯勍%囉玉萍%Douglas B.Lowrie
오연%마휘%범소용%곡경%라옥평%Douglas B.Lowrie
内含子%疫苗,DNA%免疫%热休克蛋白质类
內含子%疫苗,DNA%免疫%熱休剋蛋白質類
내함자%역묘,DNA%면역%열휴극단백질류
Introns%Vaccines,DNA%Immunity%Heat-shock proteins
目的 探讨以内含子A增强外源抗原在真核细胞中的表达效率及其对DNA疫苗在小鼠中免疫原性的影响.方法 以分枝杆菌热休克蛋白65 (Hsp65)为模式抗原,将其分别克隆入含有内含子A和不含内含子A的真核表达载体pCMV4.0和pVAX1,将表达Hsp65的不同重组质粒瞬时转染人胚肾上皮细胞系293T并免疫接种BALB/c小鼠,应用ELISA分析其所诱导产生的抗原特异性抗体水平及亚类,并以酶联免疫斑点试验(ELISPOT)和胞内细胞因子染色技术(ICS)分析其诱导产生的细胞免疫应答反应.结果 含有内含子A的重组表达质粒pCMV4.0hsp65较之不含内含子A的pVAX1 hsp65在293T细胞中的抗原表达量更高,免疫小鼠6周后诱导产生的抗-Hsp65特异性总IgG抗体水平(3.76 ±0.23对3.15 ±0.22,P＜0.01)和IgG2a/IgG1比值(4.08 ±0.04对2.23±0.12,P＜0.01)也更高,差异有统计学意义；分泌γ-干扰素(IFN-γ)的CD4+T淋巴细胞频率[(2.0±0.058)％对(1.5±0.087)％,t=4.804,P＜0.01]和CD8+T淋巴细胞频率[(0.6±0.058)％对(1.0±0.115)％,t=3.098,P＜0.05]增加,差异有统计学意义,提示其诱导产生了增强的Th1型免疫应答.结论 内含子A可增强分枝杆菌Hsp65在真核细胞中的表达效率并可提高DNA疫苗在小鼠中的免疫原性.
目的 探討以內含子A增彊外源抗原在真覈細胞中的錶達效率及其對DNA疫苗在小鼠中免疫原性的影響.方法 以分枝桿菌熱休剋蛋白65 (Hsp65)為模式抗原,將其分彆剋隆入含有內含子A和不含內含子A的真覈錶達載體pCMV4.0和pVAX1,將錶達Hsp65的不同重組質粒瞬時轉染人胚腎上皮細胞繫293T併免疫接種BALB/c小鼠,應用ELISA分析其所誘導產生的抗原特異性抗體水平及亞類,併以酶聯免疫斑點試驗(ELISPOT)和胞內細胞因子染色技術(ICS)分析其誘導產生的細胞免疫應答反應.結果 含有內含子A的重組錶達質粒pCMV4.0hsp65較之不含內含子A的pVAX1 hsp65在293T細胞中的抗原錶達量更高,免疫小鼠6週後誘導產生的抗-Hsp65特異性總IgG抗體水平(3.76 ±0.23對3.15 ±0.22,P＜0.01)和IgG2a/IgG1比值(4.08 ±0.04對2.23±0.12,P＜0.01)也更高,差異有統計學意義；分泌γ-榦擾素(IFN-γ)的CD4+T淋巴細胞頻率[(2.0±0.058)％對(1.5±0.087)％,t=4.804,P＜0.01]和CD8+T淋巴細胞頻率[(0.6±0.058)％對(1.0±0.115)％,t=3.098,P＜0.05]增加,差異有統計學意義,提示其誘導產生瞭增彊的Th1型免疫應答.結論 內含子A可增彊分枝桿菌Hsp65在真覈細胞中的錶達效率併可提高DNA疫苗在小鼠中的免疫原性.
목적 탐토이내함자A증강외원항원재진핵세포중적표체효솔급기대DNA역묘재소서중면역원성적영향.방법 이분지간균열휴극단백65 (Hsp65)위모식항원,장기분별극륭입함유내함자A화불함내함자A적진핵표체재체pCMV4.0화pVAX1,장표체Hsp65적불동중조질립순시전염인배신상피세포계293T병면역접충BALB/c소서,응용ELISA분석기소유도산생적항원특이성항체수평급아류,병이매련면역반점시험(ELISPOT)화포내세포인자염색기술(ICS)분석기유도산생적세포면역응답반응.결과 함유내함자A적중조표체질립pCMV4.0hsp65교지불함내함자A적pVAX1 hsp65재293T세포중적항원표체량경고,면역소서6주후유도산생적항-Hsp65특이성총IgG항체수평(3.76 ±0.23대3.15 ±0.22,P＜0.01)화IgG2a/IgG1비치(4.08 ±0.04대2.23±0.12,P＜0.01)야경고,차이유통계학의의；분비γ-간우소(IFN-γ)적CD4+T림파세포빈솔[(2.0±0.058)％대(1.5±0.087)％,t=4.804,P＜0.01]화CD8+T림파세포빈솔[(0.6±0.058)％대(1.0±0.115)％,t=3.098,P＜0.05]증가,차이유통계학의의,제시기유도산생료증강적Th1형면역응답.결론 내함자A가증강분지간균Hsp65재진핵세포중적표체효솔병가제고DNA역묘재소서중적면역원성.
Objective To explore the involvement of intron A into eukaryotic expression vector to improve antigen expression efficiency and enhance immunogenicity of DNA vaccine in mice.Methods As model antigen,the coding gene of mycobacterial Hsp65 was cloned into eukaryotic expression vector pCMV4.0 with intron A involved and pVAX1 without intron A involved,respectively.The resulted recombinant expression vectors were transfected into 293T cells and were then injected into BALB/c mice as DNA vaccines.Anti-Hsp65 specific IgG and isotype were detected by ELISA and T cell immune response was analyzed by enzyme-linked immunosorbent spot ( ELISPOT ) assay and intracellular cytokine staining.Results Compared with non-intron A pVAX1hsp65,the recombinant plasmid pCMV4.0hsp65 involved with intron A pVAXlhsp65 caused higher expression level of Hsp65 in 293T cells,and enhanced Thl type immune response,which was defined as higher level of anti-Hsp65 specific total IgG level (3.76 ±0.23 vs 3.15 ±0.22,P＜0.01) and IgG2a/IgG1 ratio (4.08 ±0.04 vs 2.23 ±0.12,P＜0.01) and more IFN-γ-secreting CD4+ ((2.0±0.058)％ vs (1.5 ±0.087)％,t=4.804,P＜0.01) and CD8+ ((0.6 ± 0.058) ％ vs ( 1.0 ± 0.115 ) ％,t =3.098,P ＜ 0.05 ) T lymphocytes.The difference showed statistical significance.Conclusion Intron A can improve the expression efficiency of mycobacterial Hsp65 antigen and enhance immunogenicity of DNA vaccine in mice when involved into eukaryotic expression vector.
