CAJ | 학술논문

目的分析黏膜佐剂大肠埃希菌不耐热肠毒素B亚单位(LTB)辅佐的淋病奈瑟菌孔洞蛋白B(PorB)核酸疫苗诱导小鼠的免疫应答水平.方法 PCR法扩增目的基因porB、ltB、ltB-porB,分别构建3种相应的peDNA3.1(-)真核重组表达载体,经PCR、双酶切及基因测序鉴定后转染Hela细胞,用细胞免疫荧光法鉴定质粒的蛋白表达.将核酸疫苗经鼻饲免疫雌性BALB/c小鼠,检测体液免疫及细胞免疫应答水平,同时用免疫组织化学法检测porB、ltB、ltB-porB基因在小鼠鼻黏膜内的表达.组间均数比较采用方差分析.结果构建的真核重组质粒均能在Hela细胞内及小鼠鼻黏膜组织内表达.核酸疫苗免疫组小鼠的生殖道灌洗液PorB特异性sIgA及血清porB特异性IgG水平明显高于对照组(P＜0.01;P＜0.05),且ltB-porB融合基因组特异性sIgA明显高于porB组(P＜0.05);pcDNA3.1(-)/porB组小鼠脾淋巴细胞培养上清液中IFN-γ、IL-4和脾淋巴细胞刺激指数(SI)分别为(170.04±23.89)pg/mL、(114.68±14.27)pg/mL和1.68±0.19,pcDNA3.1(-)/ltB-porB组分别为(161.42±27.50)pg/mL、(124.16±19.04)pg/mL和1.73±0.28,均高于对照组pcDNA3.1(-)和PBS,差异均有统计学意义(P＜0.01;P＜0.05),高于对照组pcDNA3.1(-)/ltB,差异均有统计学意义(均P＜0.05),但两核酸疫苗免疫组间差异无统计学意义(均P＞0.05).结论所构建的核酸疫苗分别在Hela细胞及小鼠鼻黏膜组织内获得了表达,经黏膜途径免疫能诱导小鼠产生较高水平的特异性体液免疫和细胞免疫应答,尤其是黏膜免疫应答;证实黏膜佐剂LTB可辅佐PorB诱导小鼠产生高水平的生殖道黏膜免疫.
목적 분석점막좌제대장애희균불내열장독소B아단위(LTB)보좌적임병내슬균공동단백B(PorB)핵산역묘유도소서적면역응답수평.방법 PCR법확증목적 기인porB、ltB、ltB-porB,분별구건3충상응적peDNA3.1(-)진핵중조표체재체,경PCR、쌍매절급기인측서감정후전염Hela세포,용세포면역형광법감정질립적단백표체.장핵산역묘경비사면역자성BALB/c소서,검측체액면역급세포면역응답수평,동시용면역조직화학법검측porB、ltB、ltB-porB기인재소서비점막내적표체.조간균수비교채용방차분석.결과 구건적진핵중조질립균능재Hela세포내급소서비점막조직내표체.핵산역묘면역조소서적생식도관세액PorB특이성sIgA급혈청porB특이성IgG수평명현고우대조조(P＜0.01;P＜0.05),차ltB-porB융합기인조특이성sIgA명현고우porB조(P＜0.05);pcDNA3.1(-)/porB조소서비림파세포배양상청액중IFN-γ、IL-4화비림파세포자격지수(SI)분별위(170.04±23.89)pg/mL、(114.68±14.27)pg/mL화1.68±0.19,pcDNA3.1(-)/ltB-porB조분별위(161.42±27.50)pg/mL、(124.16±19.04)pg/mL화1.73±0.28,균고우대조조pcDNA3.1(-)화PBS,차이균유통계학의의(P＜0.01;P＜0.05),고우대조조pcDNA3.1(-)/ltB,차이균유통계학의의(균P＜0.05),단량핵산역묘면역조간차이무통계학의의(균P＞0.05).결론 소구건적핵산역묘분별재Hela세포급소서비점막조직내획득료표체,경점막도경면역능유도소서산생교고수평적특이성체액면역화세포면역응답,우기시점막면역응답;증실점막좌제LTB가보좌PorB유도소서산생고수평적생식도점막면역.
Objective To investigate the specific humoral immune response and cellular immune response induced by DNA vaccine with Neisseria gonorrhoeae porin B (PorB) fused with B subunit of Escherichia coli heat-labile enterotoxin B (LTB) in mice. Methods Target genes of porB, ltB and ltB-porB were amplified by polymerase chain reaction (PCR) and cloned into eukaryotic vector pcDNA3.1(-). The recombinants were identified by PCR, enzyme digestion and DNA sequencing.The vectors were transfected into Hela cells, and expressed proteins were checked by cytoimmunofluorescence. Female BALB/c mice were intranasally immunized with recombination vectors. The humoral immune response and cellular immune response were detected by enzyme linked immunosorbent assay (ELISA) and methyl thiazolyl tetrazolium (MTT) colorimetric assay. The expressions of recombination vectors in intranasal mucosal tissues of the immunized mice were detected by immunohistochemistry. The means between groups were compared by analysis of variance. Results All the three recombinants were expressed in Hela cells and intranasal mucosal tissues. The PorB specific IgG in serum and sIgA in vaginal secretions in DNA vaccine immunized mice were significantly higher than those in controls (P＜0.01 ; P＜0.05). Moreover, the sIgA level in pcDNA3.1 (-)/ltB-porB group was higher than that in peDNA3, 1(-)/porB group (P＝0. 002). The levels of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) in the supernatants and stimulation index (SI) of spleen lymphocyte culture in pcDNA3, 1(-)/porB group were (170.04±23.89) pg/mL, (114.68±14.27) pg/mL and 1. 68±0.19, respectively; and those in pcDNA3, 1(-)/ltB-porB group were (161.42±27.50) pg/mL, (124.16±19.04) pg/mL and 1.73±0.28, respectively; which were both higher than those in pcDNA3.1(-)/ phosphate buffered saliae (PBS) group (P＜0. 01; P＜0.05) and pcDNA3.1 (-)/ltB group (all P＜0.05), while there was no significant difference between pcDNA3.1 (-)/ltB-porB group and pcDNA3. 1 (-)/porB group (0. 998, 0. 696, 0. 994; all P＞0.05). Conclusions The constructed DNA vaccines are all successfully expressed in Hela cells and murine intranasal mucosal tissues. The mucosal immunization of the vaccines [pcDNA3. 1 (- )/porB and pcDNA3.1 ( -)/ltBporB] could induce humoral immune response and cellular immune response, especially mucosal immune response. It is confirmed that mucosal adjuvant LTB could promote PorB to induce higher level of mucosal immune response in mice.