葡糖基转移酶多肽偶联疫苗抗原性

目的：研究人工合成葡糖基转移酶GTF第552—570位的多肽疫苗HDS的免疫原性。方法：将HDS与大分子裁体钥孔帽贝血蓝蛋白偶联后，在腮腺区皮下免疫大鼠，取鼠血清，Western blot法检测鼠血清抗GTT抗体。结果：硝酸纤维膜上43KD-66KD间出现强阳性条带，此条带位置与葡糖基转移酶经SDS-PAGE电泳后条带位置一致。结论：抗人工抗原HDS-KLH抗体能与葡糖基转移酶发生抗原抗体反应，抗HDS-SLH抗体具有识别葡糖基转移酶的能力。     

GTF-PAc融合防龋DNA疫苗研究(Ⅲ)pGLUA-P免疫定菌鼠防龋研究

目的 :了解GTF -PAc融合防龋DNA疫苗 pGLUA -P激发机体免疫反应和抑制龋病的能力。 方法 :将pGLUA -P和携带 pac基因A -P基因片段的DNA防龋疫苗 pCIA -P以颌下腺周围区域皮下注射 (TSG)和股四头肌注射途径分别免疫定菌SD大鼠 ,以ELISA法检测血清和唾液中的抗体水平 ,采用Keyes法评估大鼠磨牙患龋情况。结果 :pGLUA -P和 pCIA -P经TSG免疫及 pGLUA -P经股四头肌注射免疫组的血清抗PAc的IgG抗体水平明显高于对照组 (P <0 .0 5 ) ,pGLUA -P和pCIA -P经TSG免疫组的唾液抗PAc的IgA抗体水平明显高于其余组 (P <0 .0 5 ) ;pGLUA -P经TSG免疫组的釉质龋和牙本质浅龋记分最低 (P <0 .0 5 ) ;经TSG免疫 pGLUA-P组和pCIA -P组的牙本质中龋记分最低 (P <0 .0 5 )。结论 :GTF -PAc融合防龋DNA疫苗pGLUA -P可以有效地诱导机体的免疫反应 ,抑制龋病的发生和发展 ,防龋效果优于防龋DNA疫苗 pCIA -P。     

GTF—PAc融合防龋DNA疫苗研究（Ⅲ）pGLUA—P免疫定菌鼠防龋研究

目的；了解GTF－PAc融合防龋DNA疫苗pGLUA-P激发机体免疫反应和抑制龋病的能力。方法：将pGLUA-P和携带pac基因A－P基因片段的DNA防龋疫苗pCIA-P以颌下腺周围区域皮下注射（TSG）和股四头肌注射途径分别免疫定菌SD大鼠，以ELISA法检测血清和唾液中的抗体水平，采用Keyes法评估大鼠磨牙患龋情况。结果：pGLUA-P和pCIA-P经TSG免疫及pGLUA-P经股四头肌注射免疫组的血清抗PAc的IgG抗体水平明显高于对照组（P＜0．05），pGLUA-P和pCI-AP经TSG免疫组的唾液抗PAc的IgA抗体水平明显高于其余组（P＜0．05）；pGLUA-P经TSG免疫组的釉质龋和牙本质浅龋记分最低（P＜0．05）；经TSG免疫pGLUA-P组和pCIA-P组的牙本质中龋记分最低（P＜0．05）。结论：GTF－PAc融合防龋DNA疫苗pGLUA-P可以有效地诱导机体的免疫反应，抑制龋病的发生和发展，防龋效果优于防龋DNA疫苗pCIA-P。     

变形链球菌防龋基因疫苗pcDNA3-gtfB免疫途径筛选的实验研究

目的:利用含有葡糖基转移酶抗原基因的重组质粒pcDNA3-gtfB作为基因疫苗,筛选有效的免疫途径。方法:重组质粒pcDNA3-gtfB通过股四头肌注射、鼻腔灌注和颌下腺周注射免疫Wistar大鼠,采用ELISA法测定血清 IgG、唾液IgA的动态变化。结果:经股四头肌注射免疫后产生的血清IgG抗体水平明显高于其它两组血清IgG抗体水平(P<0101),且鼻腔灌注组和颌下腺周注射组间的血清IgG抗体水平无显著性差异(P>0105)。各组唾液S-IgA 抗体水平均存在显著差异(P<0101),腺周注射组产生的唾液S-IgA抗体水平最高(P<0101)。结论:基因疫苗通过腺周注射免疫途径能最有效激发特异性唾液S-IgA抗体的产生,可望成为一种有效的防龋基因疫苗免疫途径,为下一步抗龋动物实验提供了实验基础。     

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。     

人工合成多肽防龋疫苗HDS的研究--Ⅰ多肽HDS序列合成和鉴定

目的人工合成变形链球菌葡糖基转移酶第549～567位多肽序列HDS.方法应用固相多肽合成仪合成变形链球菌葡糖基转移酶上549～567位氨基酸序列HDS,使用基质辅助激光解吸电离飞行时间质谱法碎片结构分析技术,对合成产物进行检测.结果准确合成了19个氨基酸的多肽序列HDS,纯度达97%.结论成功合成变形链球菌葡糖基转移酶多肽片断为多肽防龋疫苗研究提供了基础.     

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。     

变形链球菌GbpA的GBD基因疫苗动物免疫防龋研究

目的：研究变形链球菌GbpA的GBD基因疫苗免疫SD大鼠时,其所诱导的血清和唾液特异性免疫反应及防龋效果。方法：实验分2组,实验组用纯化的变形链球菌GbpA的GBD真核表达质粒pcDNA3．1-GBD颌下腺周围皮下免疫SD大鼠,其中实验1组免疫后间隔一定的时间收集大鼠的血清及唾液,间接ELISA检测血清及唾液中特异性抗GBD抗体,实验2组免疫同时喂致龋饲料—Keyes改良的高糖Diet 2000,并于第一次注射20d时连续3d大鼠口腔中接种S．mutans Ingbritt,在接种S．mutans后第77 d处死大鼠,收集大鼠颌骨标本用于龋齿记分分析。对照组2只大鼠颌下腺周皮下注射1×PBS(pH7．4)。结果：用纯化的pcDNA3．1-GBD基因疫苗免疫大鼠,血清中IgG及唾液中IgA明显升高,大鼠高糖饮食及口腔接种S．mutans Ingbritt,实验组的龋患率明显低于对照组,说明GBD基因疫苗具有免疫防龋作用。结论：变形链球菌GbpA的GBD基因疫苗具有免疫原性,是一种有效的免疫防龋疫苗。     

*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。     

葡糖基转移酶合成肽疫苗的免疫原性研究

目的　检测合成的葡糖基转移酶 (glucosyltransferase ,GTF)多肽疫苗的免疫原性 ,有助于研制以合成多肽为基础的防龋疫苗。方法　合成融有GTF催化和葡聚糖结合区的 2 7个氨基酸残基肽段 ,利用ELISA法检测免疫小鼠抗体的产生 ,通过GTF酶活性与变形链球菌粘附实验测定其抗血清的作用。结果　融合多肽疫苗的序列为ANDVDNSNPVVQAEQLYFRANGVQVKG ,免疫小鼠脾脏重量显著增加 ,可有效诱导机体抗体的产生。其免疫血清不仅拮抗GTF的酶活性 ,而且明显抑制变形链球菌的粘附。结论　GTF多肽疫苗可产生抗体介导的抑制GTF酶活性和抑制葡聚糖结合作用 ,对龋病的防治十分有价值     

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     

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.     

Properties of practical oral liposome-Streptococcus mutans glucosyltransferase vaccines for effective induction of caries protection

Here we report the effectiveness of various liposome vaccines containing Streptococcus mutans glucosyltransferase (GTF) in protecting against dental caries after oral immunization. Rats were immunized by gastric intubation of the appropriate liposome vaccine at weaning and boosted 3 times. Rats were infected with S. mutans following initial immunization and fed cariogenic diet (Diet 305). Saliva and serum were collected during the study and assessed for antibody activity by enzyme-linked immunosorbent assay. Mandibles were removed on day 47 and assessed for S. mutans levels and then for caries. Animals immunized with sonicated, filtered and microemulsined GTF liposome preparations had decreased levels of dental caries compared with control animals given empty liposomes. Rats given dehydrated/rehydrated or purified liposomal GTF also had significantly less caries than control group (GTF alone). Because of economy, ease of preparation and efficiency in amount of antigen used, filtered, dehydrated/rehydrated and purified liposomal GTF preparations are most practical for use in further assessing the efficacy of liposomal GTF in oral immunization.     

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.     

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     

变形链球菌GbpA的GBD免疫防龋实验研究

目的：观察变形链球菌GbpA的GBD融合蛋白免疫SD大鼠的防龋效果。方法：用纯化的变形链球菌GbpA的GBD融合蛋白皮下免疫SD大鼠,喂Keyes改良的高糖Diet 2000,第1次免疫20d时,连续3d大鼠口腔中接种S．mutans Ingbritt,在接种S．mutans后第77天处死大鼠,收集大鼠颌骨标本,用于龋齿记分分析,用t检验进行统计学分析。结果：GBD融合蛋白免疫大鼠,实验组龋损范围及龋坏程度均显著低于对照组,P＜0．01。结论：变形链球菌GbpA的GBD融合蛋白疫苗可有效降低龋齿的发生。