阳离子脂质体乳剂增强防龋疫苗黏膜免疫效果的研究

目的：观察阳离子脂质体增强防龋疫苗诱导的小鼠特异性IgG和唾液IgA抗体水平。方法：以阳离子脂质体乳剂分别包被防龋DNA疫苗（pGJA-P/VAX）和基因重组rPAc蛋白疫苗,用DNA初免,2周后蛋白加强免疫的策略滴鼻免疫6～8周龄的BALB/c小鼠,分别收集初免后0、2、4、6、8、10、12、16周的血清和唾液,用酶联免疫吸附实验（ELISA）检测血清和唾液中特异性抗体的水平。结果：制备的乳剂疫苗CLE/DNA和CLE/rPAc的粒径分别为134.6nm和230nm,ZETA电位为36mV和18.5mV,乳剂疫苗免疫小鼠后可以诱导血清特异IgG和唾液特异IgA产生,并且实验组（LE/DNA初免＋LE/rPAc加强免疫组）能产生较对照组（DNA初免＋rPAc加强免疫组）更持久的黏膜免疫反应,两组比较差异有显著性（P〈0.05）。结论：阳离子脂质体乳剂是有效的基因疫苗载体,与防龋疫苗（DNA和rPAc蛋白）合用免疫小鼠,可增强血清特异性IgG和唾液特异性IgA抗体水平,延长抗体维持时间,具有较好黏膜免疫反应增强作用。     

中试防龋DNA疫苗pGJA-P/VAX免疫小鼠的实验研究

目的：评价中试防龋DNA疫苗pGJA-P/VAX诱导小鼠体内特异性免疫应答的效果。方法：分别由武汉生物制品研究所中试制备和实验室采用德国Qiagen公司去内毒素质粒大量提取试剂盒手工提取防龋DNA疫苗pGJA-P/VAX;两种方法制备的质粒分别于0周,2周经肌肉、鼻腔免疫小鼠,并以鞭毛蛋白作为黏膜佐剂与质粒同时经鼻腔免疫小鼠,PBS及空载体pVAX1作为对照。免疫前开始每两周收集小鼠血清和唾液样本,ELISA定量检测血清及唾液中特异性抗体含量。结果：中试质粒和手提质粒肌注时均能够诱导血清特异性IgG,但诱导唾液特异性sIgA的能力较为有限。在与黏膜佐剂鞭毛蛋白同时滴鼻时,中试质粒可诱导较为明显的血清特异性IgG和唾液特异性sIgA。在相同的免疫条件下,中试质粒诱导的特异性抗体水平低于手提质粒组,但二者无显著性差异。结论：中试防龋DNA疫苗pGJA-P/VAX能够诱导小鼠体内特异性体液免疫应答。     

A DNA vaccine encoding a cell-surface protein antigen of Streptococcus mutans protects gnotobiotic rats from caries

A cell-surface protein antigen (PAc) of Streptococcus mutans is considered a virulence factor because it may mediate initial attachment of Streptococcus mutans to tooth surfaces. Thus, inhibiting PAc is predicted to provide protection against caries. To develop vaccines against dental caries, we constructed a DNA vaccine, pCIA-P, which encodes two high-conservative regions of PAc. Expression of the recombinant protein was obtained in eukaryotic cells in vitro and in vivo. In this report, we provide evidence that fewer caries lesions, and high levels of PAc-specific salivary IgA antibody and serum IgG antibody, were observed in gnotobiotic rats following targeted salivary gland (TSG) administration of pCIA-P. This study shows that the recombinant DNA vaccine pCIA-P could induce protective anti-caries immune responses and that TSG immunization is a promising strategy for the inhibition of dental caries.     

Immunogenicity and persistence of a targeted anti-caries DNA vaccine

We have previously reported that a targeted anti-caries DNA vaccine, pGJA-P, induced accelerated and increased antibody responses compared with a non-targeted anti-caries DNA vaccine. Recently, pGJA-P/VAX, a new targeted anti-caries DNA vaccine for human trials, was constructed by replacing the pCI vector used in the construction of pGJA-P with pVAX1, the only vector authorized by the US Food and Drug Administration in clinical trials. Here, we report on our exploration of the kinetics of the antibody responses generated following pGJA-P/VAX immunization and the persistence of pGJA-P/VAX at both the inoculation site and the draining lymph nodes. Intranasal vaccination of mice with pGJA-P/VAX induced strong antibody responses that lasted for more than 6 months. Furthermore, pGJA-P/VAX could still be detected at both the inoculation site and the draining cervical lymph nodes 6 months after immunization. Thus, the persistent immune responses are likely due to the DNA depot in the host, which acts as a booster immunization.     

Immunogenicity and protective efficacy of a targeted fusion DNA construct against dental caries

Targeting antigens to antigen-presenting cells by fusion to cytotoxic T lymphocyte-associated antigen 4 (CTLA4) has been shown to be a highly efficient method to enhance the efficacy of DNA vaccines. The purpose of this study was to determine the immunogenicity and protective efficacy of the targeted fusion DNA construct pGJA-P, which contains the signal peptide and extracellular regions of human CTLA4 gene, the hinge and Fc regions of human Iggamma1 gene, the glucan-binding domain of the Streptococcus mutans gtfB gene and the A-P fragment of the S. mutans pac gene, compared with the fusion DNA construct pGLUA-P, which contains only the glucan-binding domain of the S. mutansgtfB gene and the A-P fragment of the S. mutans pac gene. BALB/c mice were immunized with pGJA-P, pGLUA-P, or pCI (vector) by the intramuscular or intranasal route. Specific anti-PAc and anti-GTF-I serum IgG and salivary IgA antibody responses were assessed by an enzyme-linked immunosorbent assay. Wistar rats were orally challenged with S. mutans and immunized with pGJA-P, pGLUA-P, or pCI intramuscularly or intranasally, and caries activity was evaluated by the Keyes method. pGJA-P induced accelerated and increased serum and salivary antibody responses in mice compared with pGLUA-P. Rats immunized with pGJA-P had significantly fewer caries lesions than rats immunized with pGLUA-P (p < 0.01). Thus, this study demonstrates that the targeted DNA construct pGJA-P can enhance both systemic and mucosal immunity and may be a useful strategy for improving the protective efficacy of anticaries DNA vaccines.     

靶向融合防龋DNA疫苗pGJA-P免疫兔的实验研究

目的　体外检测靶向融合防龋DNA疫苗pGJA P的免疫反应性 ;与非靶向融合防龋DNA疫苗 pGLUA P进行比较 ,评价其增强疫苗免疫效能的能力。 方法　将 pGJA P转染CHO细胞 ,蛋白质免疫印迹实验检测重组蛋白抗体免疫反应性。分 5组免疫兔 :pGJA P经肌肉注射组 (A组 ) ;pGJA P经鼻黏膜免疫组 (B组 ) ;pGLUA P经肌肉注射 (C组 ) ;pGLUA P经鼻黏膜免疫组 (D组 )和pCI载体经股四头肌注射组 (E组 )。酶联免疫吸附实验检测血清及唾液中的特异性抗体。用收集的血清进行葡糖基转移酶 (GTF)合成水不溶性葡聚糖抑制实验。结果　pGJA P表达的重组蛋白可以与抗GTF抗体反应。A组血清特异性IgG抗体水平远高于C组 (P <0 0 1) ;B组唾液特异性IgA抗体水平显著高于D组 (P <0 0 1) ;A组同样诱导了高水平的唾液特异性IgA抗体。A组的免疫血清抑制GTF活性的能力最强。结论　pGJA P具备GTF的免疫反应性 ;并较pGLUA P有更强的诱导系统和黏膜免疫反应及抑制GTF的能力。     

DNA防龋疫苗不同途径免疫BALB/c小鼠的实验研究

目的：观察编码pac结构基因A—P片段的重组质粒pCIA—P以不同途径免疫BALB／c小鼠后的特异性免疫反应。方法：重组质粒pCIA—P经鼻腔滴注和颌下腺区皮下腺注射两种途径免疫小鼠，ELISA法检测血清和唾液中特异性抗体水平动态变化。结果：滴鼻法和皮下注射法免疫后唾液IgA型特异性抗体和血清IgG型特异性抗体均明显升高，并持续数周。且滴鼻法比皮下注射法诱导唾液IgA型特异性抗体要早。结论：重组质粒pCIA—P是一种有效的免疫原，滴鼻法和颌下区皮下注射法接种防龋DNA疫苗都可有效诱导机体全身及局部黏膜免疫应答。而滴鼻法较皮下注射法能更早诱导局部黏膜免疫应答。     

靶向融合防龋DNA疫苗免疫大鼠的研究

目的　检测靶向融合防龋DNA疫苗 pGJA P免疫大鼠后的原位表达。比较 pGJA P与融合防龋DNA疫苗pGLUA P免疫定菌鼠后产生的抗体水平和防龋效果。方法　质粒pGJA P分别经股四头肌注射和鼻腔滴注免疫大鼠 ,免疫组化法检测重组蛋白在免疫部位的表达。建立定菌鼠模型 ,质粒pGJA P和 pGLUA P分别经股四头肌注射和鼻腔滴注免疫定菌鼠 ,ELISA法检测血清和唾液中的特异性抗体水平 ,取上下颌骨进行Keyes龋齿记分。 结果　在 pGJA P肌肉免疫组的股四头肌和鼻腔免疫组的鼻腔黏膜检测到了表达的重组蛋白。pGJA P肌肉免疫组的血清抗PAc和抗GTFIgG滴度显著高于其他组 (P <0 0 1)。pGJA P肌肉免疫组和 pGJA P鼻腔免疫组的唾液抗PAc和抗GTFIgA滴度显著高于其他组 (P <0 0 1)。pGJA P免疫组的龋齿记分显著低于其他组 (P <0 0 1)。结论　pGJA P在动物体内能够正确表达。与pGLUA P相比 ,pGJA P能诱导更强的体液免疫反应 ,防龋效果更好     

靶向融合防龋DNA疫苗免疫田鼠的实验研究

目的 :以定菌田鼠为动物模型 ,经不同免疫途径 ,比较 3种防龋DNA疫苗 [pGLUA -P(以 pCI为载体 ) ,pGJA -P(以 pCI为载体 ) ,pGJA -P(以 pVAX为载体 ) ]的免疫防龋效果。 方法 :将 96只田鼠随机分为 12组 ,接种变形链球菌并给予致龋饲料 ,按 6因素 (3种质粒 ,2种空载体 ,生理盐水 ) 2水平 (肌肉 ,粘膜途径 )析因设计方案免疫田鼠 ,采用ELISA方法检测各组田鼠血清IgG和唾液IgA水平 ,参照Keyes经典计分标准进行龋齿计分 ,观察各组田鼠磨牙患龋情况和一般安全性指标。结果 :疫苗免疫组特异性抗Pac血清IgG和唾液IgA抗体水平均显著高于非疫苗免疫组 (P <0 .0 1) ,而龋齿记分显著低于非疫苗免疫组 (P <0 .0 1)。同一接种途径中 ,不同质粒刺激机体产生的抗体水平不同 ,同种质粒 ,经不同途径接种诱导机体产生抗体水平也不同 ,其中以鼻粘膜免疫接种pGJA-P(以 pCI为载体 ) ,pGJA -P(以pVAX为载体 )组的唾液抗Pac的sIgA水平高 ,龋齿记分低 ,与其它实验组比较差异有显著性 (P <0 .0 1)。结论 :靶向融合防龋DNA疫苗能够激发机体产生免疫反应 ,经粘膜免疫可诱导较高的唾液抗Pac的sIgA ,并且有效的控制龋齿的发生和发展。     

靶向防龋DNA疫苗在小鼠体内的存留时间分析

目的：观察靶向防龋DNA疫苗pG JA-P/VAX免疫小鼠后在免疫原位和引流淋巴结的存留时间。方法：靶向防龋DNA疫苗pG JA-P/VAX分别经肌肉注射和鼻黏膜滴注途径免疫BALB/c小鼠。免疫后1、3、6个月,取肌肉注射组注射局部的肌肉组织和腹股沟引流淋巴结,鼻黏膜滴注组鼻相关淋巴组织和颈部引流淋巴结,提取基因组DNA,以其为模板采用PCR方法扩增pG JA-P/VAX中的GLU序列。结果：免疫后1、3、6个月,在免疫局部组织和引流淋巴结的基因组DNA中均扩增得到870 bp的片段,测序证实该片段序列与GLU序列一致。结论：防龋DNA疫苗pG JA-P/VAX免疫小鼠后,可在免疫原位和引流淋巴结较长期存在。     

细胞毒淋巴细胞相关抗原4靶向防龋DNA疫苗在细胞中的表达研究

目的采用绿色荧光蛋白标记技术,研究细胞毒淋巴细胞相关抗原4（CTLA-4）靶向DNA疫苗编码抗原在细胞中表达的一般形式,并探讨CTLA-4融合抗原片段大小对靶向DNA疫苗在细胞中表达水平的影响.方法从靶向融合防龋DNA疫苗pGJA-P中去掉A-P片段,构建出靶向防龋质粒pGJGLU;以pVAX1质粒骨架替代pGJA-P和pGJGLU质粒中的pCI载体骨架,构建出pGJA-P/VAX和pGJLU/VAX真核表达质粒;切下pGJGLU质粒的CTLA-4-Ig-GLU片段,克隆入pEGFP-N1中,获得pGJGLU/GFP质粒,转染CHO细胞,荧光显微镜下观察蛋白表达;将pGJA-P/VAX、pGJGLU/VAX和pGJGLU/GFP转染CHO细胞,以ELISA法检测培养上清液中融合蛋白的表达水平.结果pGJGLU/GFP转染细胞镜下表达特异性绿色荧光物质,呈颗粒状;pGJA-P/VAX、pGJGLU/VAX和pGJGLU/GFP 3种质粒可以表达融合蛋白并分泌到培养上清液中,且所表达的融合蛋白浓度不同,并以pGJGLU/VAX表达的水平最高.结论CTLA-4融合蛋白可以以分泌形式表达,CTLA-4靶向疫苗编码的融合抗原片段大小影响其在细胞中的表达分泌水平.     

两种GTF氨基催化区改良防龋质粒免疫小鼠的实验研究

目的：通过比较编码表兄链球菌葡糖基转移酶氨基催化区CAT不同形式基因序列的两种改良防龋质粒免疫BALB/c小鼠后激发的特异性抗体水平差异,初步评价不同形式CAT在DNA防龋疫苗中的应用潜力。方法：利用分子克隆技术构建两种改良防龋质粒,在靶向防龋DNA疫苗pGJA-P/VAX中插入表兄链球菌OMZ176GTF-I CAT全基因序列构建pGJGAC/VAX;将基因公司合成的共计165bp的串联重复5次的GTF-I氨基催化区核心保守序列克隆到pGJA-P/VAX中构建pGJGA-5C/VAX。转染CHO细胞系,检测其表达。两种改良质粒经鼻腔滴注免疫BALB/c小鼠,检测血清和唾液中的特异性抗PAc,抗GLU和抗CAT抗体水平。结果：重组质粒的基因序列与预期相符。不同形式CAT基因序列的两种改良防龋质粒均可在真核细胞正确表达。pGJGAC/VAX和pGJGA-5C/VAX免疫动物后血清和唾液特异性抗PAc、抗GLU和抗CAT抗体均显著高于空载体pVAX1免疫组（P〈0.05）。第10、12和14周,pGJGAC/VAX免疫组小鼠的血清特异性抗CAT抗体显著高于pGJGA-5C/VAX组（P〈0.05）。两组间唾液特异性抗体未见显著性差异。pGJGA-5C/VAX在小鼠体内激发特异性抗体的速度较pGJGAC/VAX略快。结论：本实验构建的增加表兄链球菌GTF氨基催化区的两种改良防龋质粒,可在真核细胞中正确表达,两种质粒有效地诱导黏膜和系统体液免疫反应,pGJGAC/VAX和pGJGA-5C/VAX各有优势。     

DNA防龋疫苗联合蛋白质疫苗诱导小鼠免疫应答的研究

目的:研究以一种新型免疫策略即追加相应蛋白质抗原来加强免疫DNA疫苗的免疫效果。方法:重组质粒pCIA-P经鼻腔滴注途径免疫小鼠,并以其相应抗原rPAc蛋白或rPAc蛋白及黏膜佐剂rCTB经鼻腔滴注加强免疫,ELISA法检测血清和唾液中特异性抗体水平。结果:以rPAc蛋白或rPAc蛋白及黏膜佐剂rCTB加强免疫可显著提高唾液中IgA型特异性抗体和血清IgG型特异性抗体水平。并且以rPAc蛋白和黏膜佐剂rCTB加强免疫组产生了最高水平的唾液IgA型特异性抗体和血清IgG型特异性抗体。结论:DNA防龋疫苗联合蛋白质疫苗可有效增强DNA防龋疫苗免疫效果。     

A Caries Vaccine? The state of the science of immunization against dental caries

Studies performed in numerous laboratories over several decades have demonstrated the feasibility of immunizing experimental rodents or primates with protein antigens derived from Streptococcus mutans or Streptococcus sobrinus against oral colonization by mutans streptococci and the development of dental caries. Protection has been attributed to salivary IgA antibodies which can inhibit sucrose-independent or sucrose-dependent mechanisms of streptococcal accumulation on tooth surfaces according to the choice of vaccine antigen. Strategies of mucosal immunization have been developed to induce high levels of salivary antibodies that can persist for prolonged periods and to establish immune memory. Studies in humans show that salivary antibodies to mutans streptococci can be induced by similar approaches, and that passively applied antibodies can also suppress oral re-colonization by mutans streptococci. Progress towards practical vaccine development requires evaluation of candidate vaccines in clinical trials. Promising strategies of passive immunization also require further clinical evaluation.     

Remote glucosyltransferase-microparticle vaccine delivery induces protective immunity in the oral cavity

Intranasally administered dental caries vaccines show significant promise for human application. Alternate mucosal routes may be required, however, to induce caries-protective salivary IgA antibody in children with respiratory diseases. Since rectal mucosa contains inductive lymphoid tissue, we hypothesized that the rectal route could be used to induce salivary immunity to mutans streptococcal glucosyltransferase (GTF), resulting in protective immunity to experimental dental caries. We first explored the ability of glucosyltransferase, incorporated into polylactide-co-glycolide (PLGA) microparticles (MP), and administered rectally together with mucosal adjuvant, to induce a salivary IgA antibody response. Groups of Sprague-Dawley rats (6/group) were immunized rectally on days 0, 7, 14 and 21 with a) GTF-MP alone, b) GTF-MP with cholera toxin, c) GTF-MP with detoxified mutant Escherichia coli toxin (dLT), or d) sham immunized with PLGA and cholera toxin. An additional group was immunized intranasally with GTF-MP alone. Saliva and nasal washes of all intranasally immunized rats contained IgA antibody to glucosyltransferase on day 28. Salivary IgA antibody was also detected in 7/12 rats rectally immunized with GTF-MP and cholera toxin or dLT, although responses were lower than those obtained by intranasal immunization. Most fecal extracts from rectally delivered GTF-MP plus cholera toxin or dLT rats contained IgA antibody to GTF-MP. Low levels of fecal IgA antibody were detected in 3/6 intranasally immunized rats and 2/6 rats rectally immunized with GTF-MP alone. We then examined the extent to which salivary IgA antibody induced by the rectal route could be protective. At 25, 31 and 38 days of age, two groups of female Sprague-Dawley rats (13/group) were rectally immunized with GTF-MP and cholera toxin or with empty microparticles and cholera toxin (sham group). A third group was intranasally immunized with GTF-MP alone. After demonstrating salivary IgA responses to GTF in most GTF-immunized rats, all animals were infected with streptomycin-resistant Streptococcus sobrinus and placed on diet 2000. After 79 days of infection, total caries on molar surfaces were lower in both rectally (7.9 +/- 1.0) and intranasally (7.1 +/- 0.9; P < 0.0.03) immunized groups compared with the sham-immunized group (11.9 +/- 1.6). Smooth surface caries were significantly lower (P < 0.05) in both rectally and intranasally immunized groups. These results support the interconnectedness of the mucosal immune system and indicate that rectal immunization with GTF-MP, together with adjuvant, or intranasal immunization with GTF-MP alone, can induce protective levels of salivary antibody in rats.     

AddaVax经不同免疫途径对防龋疫苗的佐剂效应

目的：研究3种不同免疫途径下,防龋亚单位疫苗联合AddaVax佐剂接种小鼠所产生的免疫效果。方法：以rPAc与AddaVax混合物经鼻腔滴注、颊黏膜下注射及颈背部皮下注射等3种途径免疫小鼠,并以rPAc行鼻腔滴注免疫,ELISA法检测血清和唾液中的特异性抗体水平。结果：rPAc＋AddaVax皮下及颊黏膜下注射方式均可显著提高小鼠血清中抗PAcIgG水平;rPAc＋AddaVax颊黏膜下注射方式可有效提高小鼠唾液中抗PAcIgA水平。结论：rPAc与AddaVax联合经皮下及颊黏膜下注射方式可有效提高防龋疫苗的免疫效果。     

基因重组乳链球菌防龋疫苗的研究Ⅶ.基因重组乳链球菌免疫BALB/c鼠的实验研究

目的：观察基因重组乳链球菌的免疫原性和免疫反应性。方法：对实验动物ＢＡＬＢ／ｃ鼠进行基因重组乳链球菌防龋疫苗的灌胃免疫，并以酶联免疫吸附实验进行鼠血清和唾液中抗特异性ＰＡｃ－ＩｇＧ测定。结果：接种重组乳链球菌ＨＬ１０２、ＨＬ１０７组血清中抗ＰＡｃ－ＩｇＧ明显高于乳链球菌免疫组（Ｐ＜０．０１），与接种变形乳链球菌组产生同样的ＩｇＧ反应（Ｐ＞０．０１）。结论：基因重组乳链球菌具有变形乳链球菌表面蛋白抗原的免疫原性，能够在ＢＡＬＢ／ｃ鼠中产生特异性免疫反应和免疫表达产物抗特异性ＰＡｃ－ＩｇＧ。     

Immune response in humans to a nasal boost with Streptococcus mutans antigens

We previously reported that a Streptococcus mutans enriched-glucosytransferase (E-GTF) preparation induces an immune response following intranasal, but not tonsillar, immunization of humans. In this study, we determined whether intranasal immunization of these subjects 2 years later resulted in augmented immune responses compared to those seen in control subjects. Subjects previously immunized via the intranasal (IN, n = 7) or tonsillar (IT, n = 7) route and control (n = 12) subjects were immunized via the intranasal route with E-GTF. Nasal wash, saliva, and serum were collected before immunization and then weekly for 3 months after immunization. Significant (P < 0.05) mucosal and serum immunoglobulin A (IgA) anti-E-GTF responses were observed in all three groups. Nasal and serum IgA anti-E-GTF responses were significantly higher (P < 0.05) in the IN group. The salivary responses in the three groups were, in general, similar. These results indicate that intranasal immunization primes the immune system for a localized secondary response to S. mutans antigens.     

*编码Pac结构基因的DNA疫苗免疫动物的实验研究

目的：本项研究将已构建的编码pac结构基因A－P片段的重组质粒pCIA-P免疫Wistar大鼠，观察重组表面蛋白抗原PAc在大鼠体内不同组织中的原位表达以及免疫定菌鼠后的防龋效果。方法：重组质粒pCIA-P经股四头肌肌肉注射和颌下腺区皮下注射两种途径免疫大鼠，以免疫组化技术观察重组蛋白PAc在免疫部位的表达。采用股四头肌肌肉注射，颌下腺区皮下注射和颊粘膜下注射3种方法免疫定菌鼠，ELISA法检测血清和唾液中特异性抗体水平，Keyes计分法评估免疫定菌鼠后鼠磨牙的患龋情况。结果：大鼠股四头肌细胞中可见PAc蛋白不均匀的受限表达，双侧颌下腺组织均可检测到PAc蛋白的阳性染色，在导管内表达呈强阳性。用重组质粒pCIA-P经颌下腺区皮下注射和颊粘膜免疫的方法可显著增加唾液中抗PAc-IgA水平和血清中特异性抗PAc-IgG水平，重组质粒免疫定菌鼠后能显著降低定菌鼠龋损计分。特别是颌下腺区皮下和颊粘膜注射免疫组，大鼠牙本质龋的破坏程度明显低于其他组，结论：重组质粒pCIA-P是一种有效的免疫原，粘膜免疫是较理想的DNA防龋疫苗的接种途径。     

编码基因pac的DNA疫苗经鼻粘膜免疫定菌鼠的研究

目的　检测pCIA PDNA防龋疫苗经鼻粘膜免疫定菌鼠的效果 ,并对两种不同的载体系统进行对比。方法　将 30只出生后 2 0d断乳的SD雌鼠随机分为 6组 ,制备定菌模型。分别用裸DNA(A组 )、DNA Dosper复合体 (B组 )、DNA Bupivacaine复合体 (C组 )、pCI质粒 (D组 )及无菌水 (E组 )经鼻粘膜免疫大鼠 ,裸DNA股四头肌注射 (F组 )为阳性对照。 2周后加强免疫 1次。 70d鼠龄时收集唾液、血清及粪便 ,酶联免疫吸附实验检测特异性抗体 ,处死大鼠进行Keyes记分 ,单因素方差分析法分析结果。结果　B、C、F组血清特异性抗PAcIgG水平和B、C组唾液中特异性抗PAcIgA抗体水平明显高于其他组 (P <0 0 1 )。疫苗免疫组龋齿记分明显低于阴性对照组 (P <0 0 1 ) ,其中B、C组效果最好。结论　编码基因pac的pCIA PDNA防龋疫苗经鼻粘膜途径进行免疫可以有效预防龋病的发生 ,阳离子脂质体Dosper和局部麻醉药Bupivacaine能够增强核酸疫苗的免疫效能