变异链球菌、表兄链球菌复合防龋DNA疫苗的研制

Protective efficacy of a new fusion anti-caries DNA vaccine encoding antigens of both Streptococcus mutans and Streptococcus sobrinus

目的构建包含变异链球菌和表兄链球菌两种致龋菌的主要抗原片段的复合防龋DNA疫苗，以期增强DNA防龋疫苗对表兄链球菌的抑制作用。方法PCR获得表兄链球菌OMZ176GTF—I的CAT区，克隆至靶向防龋DNA疫苗pGJA—P／VAX中，构建编码变异链球菌PAc、GLU基因序列和表兄链球菌CAT基因序列的复合防龋DNA疫苗pGJGAC／VAX，转染CHO细胞系检测其表达。重组质粒及对照质粒分别经股四头肌注射和鼻腔滴注免疫BALB／c小鼠，ELISA法检测血清和唾液中的特异性抗PAc，抗GLU和抗CAT抗体水平。重组质粒及对照质粒经鼻腔滴注免疫分别定植了变异链球菌和表兄链球菌的Wistar大鼠，龋齿记分评价防龋疫苗的龋齿保护效能。结果重组质粒pGJGAC／VAX构建成功。免疫小鼠后实验组小鼠血清和唾液抗PAc、抗GLU和抗CAT抗体水平均显著高于空载体对照组（P〈0．01）。血清特异性抗CAT抗体水平最高值在肌肉免疫组第8周和黏膜免疫组第10周时出现，分别为62．13μg/ml和11．43μg／ml；唾液特异性抗CAT抗体水平最高值在肌肉免疫组第8周和黏膜免疫组第10周时出现，分别为0．67％和0．80％。大鼠龋齿保护实验结果显示在变异链球菌和表兄链球菌定菌鼠中实验组釉质龋（E），牙本质浅龋（Ds）和牙本质中龋（Dm）均显著低于pVAX1免疫组（P〈0．05），实验组Ds和Dm均低于pGJA—P／VAX免疫组，但两组间E差异没有统计学意义（P〉0．05）。结论复合防龋DNA疫苗构建成功，可在真核细胞中正确表达，动物实验证实能有效地诱导黏膜和系统体液免疫反应，并增强了DNA防龋疫苗对表兄链球菌的抑制作用。

Objective To construct a new fusion anti-caries DNA vaccine pGJGAC/VAX encoding antigens of both S. mutans and S. sobrinus so as to enhance the protective effect of DNA vaccine against S. sobrinus infection. Methods The CAT fragment of S. sobrinus OMZ176 gtf-I was amplified by semi-nest PCR and then inserted into the plasmid pGJA-P/VAX to construct the recombinant plasmid pGJGAC/VAX. The CHO cell was transfected and the expression of fusion protein detected using cellular immunohistochemistry and Western blot. Mice were immunized with pGJGAC/VAX and control plasmids via the intramuscular （i. m） or intranasal （i. n） routes. During the experiment, blood and saliva samples were collected at a 2-week interval for antibody assay by ELISA. Rats were orally challenged with S. mutans Ingbritt or S. sobrinus 6715 and then immunized i. n with pGJGAC/VAX, pGJA-P/VAX or pVAX1. The Keyes method was used to determine the caries activity. Results （ 1 ） CAT sequence was identical to the related sequence of gtf-I （ OMZ176 ） in GenBank. The recombinant plasmid pGJGAC/VAX encoded the genes of antigens of both S. mutans and S. sobrinus. The expressed protein could respond to specific anti- PAc, anti-GLU and anti-CAT antibodies respectively. （2） As for antibody reactions, mice in the experiment group had significantly higher levels of anti-PAc, anti-GLU and anti-CAT IgG antibodies than those in the pVAX1 group （P 〈 0. 01 ）. The peak responses of specific anti-CAT antibodies were observed at 8 weeks （GAC/i. m） and 10 weeks （ GAC/i. n ） and were approximately 62. 13 μg/ml and 11.43 μg/ml respectively. The peak responses of specific anti-CAT IgA antibodies were seen at 8 weeks （ GAC/i. m） and 10 weeks （GAC/i. n） and were approximately 0. 67% and 0. 80% respectively. （3）In the group infected with S. mutans or S. sobrinus, the pGJGAC/VAX-immunized rats showed significantly fewer E, Ds and Dm lesions than pVAXl-immunized rats （ P 〈 0. 05 ） and decreased Ds and Dm levels than pGJA-P/VAX- immunized rats （ P 〈 0. 05 ） while there was no obvious difference in E lesions between the two groups （ P 〉 0. 05）. Conclusion A new fusion anti-caries DNA vaccine pGJGAC/VAX encoding antigens of both S. mutans and S. sobrinus is constructed successfully and expressed correctly in eukaryotic cells. It induces effective mucosal and systematic humoral responses so as to provide a better protection against S. sobrinus.