摘要
目的:用防龋基因疫苗pVAX1-SG和pVAX1-SSG经肌肉注射,颌下腺周注射及鼻粘膜滴注三种不同途径来分别免疫BABL/c小鼠和SD大鼠,观察其免疫效果,并探索最佳免疫途径。
方法:本研究包括三个部分
实验一:重组质粒pVAX1-SG、pVAX1-SSG及空载体质粒pVAX1的鉴定和大量制备。先小量抽提储存的重组质粒pVAX1-SG、pVAX1-SSG及空载体质粒pVAX1。并经过限制性内切酶酶切、凝胶电泳鉴定。在鉴定正确的基础上,大量抽提重组质粒并计算其浓度和纯度,贮存以备免疫动物。
实验二:用重组质粒pVAX1-SG、pVAX1-SSG及空载体质粒pVAX1免疫BABL/c小鼠的实验研究。6周龄雄性BALB/c小鼠64只,随机分为8组,每组8只。A组:生理盐水鼻腔滴注组(空白对照组);B组:pVAX1(空载质粒)股四头肌注射组(阴性对照组);C组:pVAX1-SG双侧颌下腺区皮下注射组;D组:pVAX1-SG股四头肌注射组;E组:pVAX1-SG鼻腔粘膜注滴组;F组:pVAX1-SSG双侧颌下腺区皮下注射组;G组:pVAX1-SSG股四头肌注射组;H组:pVAX1-SSG鼻腔粘膜滴注组。每周一次,连续免疫三周,每次剂量为100μg/只(生理盐水组为100μl/只)。分别于免疫前1天和免疫后1,2,3,4周收集血清和唾液样本。至第4周取血、唾液完毕后,称重并处死小鼠,解剖分离其心、肝、脾、肺、肾等重要脏器,进行病理检查。采用酶联免疫吸附实验(间接ELISA法)检测血液和唾液中IgG和S-IgA抗体水平。
实验三:用重组质粒pVAX1-SG、pVAX1-SSG及空载体质粒pVAX1免疫SD大鼠的实验研究。出生18天的雄性SD大鼠64只,建立龋齿模型;其分组情况、免疫途径、免疫时间、病理学检查及特异性抗体的检测的方法同小鼠;样本采集从免疫开始前一天至鼠龄80天时,每周采集一次;在鼠龄80天时称重并处死动物,并取上下颌骨,进行龋损评定,根据Keyes经典评分方法进行评估。
结果:①用试剂盒提出高纯度、高浓度的重组质粒pVAX1-SG、pVAX1-SSG及空载体质粒pVAX1。②pWAX1-SG和pVAX1-SSG免疫BABL/c小鼠和SD大鼠后S-IgA型特异性抗体和IgG型特异性抗体均明显升高,并持续数周。且鼻腔滴注组和颌下腺区皮下注射组比股四头肌注射组诱导产生特异性抗体要高(P＜0.05)。③在相同途径下pVAX1-SSG诱导产生的特异性抗体比pVAX1-SG诱导产生的特异性抗体高,差异有统计学意义(P＜0.05)。④实验组与阴性对照组相比,龋损明显降低,具有统计学意义(P＜0.05)。
结论:①防龋基因疫苗pVAX1-SG和pVAX1-SSG能诱导BABL/c小鼠和SD大鼠的唾液和血液中特异性抗体的产生;②两种防龋基因疫苗经三种途径免疫后均可降低SD大鼠的龋损程度。③鼻腔粘膜免疫可能是pVAX1-SG和pVAX1-SSG最有效的免疫途径。④pVAX1-SSG比pVAX1-SG能够更好的诱导特异性抗体的产生,更能有效的减轻龋损的程度。
Objective:To investigate the efficiency of pVAX1-SG and pVAX1-SSG immunized in BABL/c mice and SD rats by different routes,and to explore the best means immune.
Methods:This study consists of three parts:
Test one:identification and preparation of recombinant plasmid pVAX1-SG1, pVAX1-SSG and plasmid pVAX1.Firstly,the plasmid of pVAX1-SG,pVAX1-SSG and PVAX1 were prepared in a small quantity by E.Z.N.A.Plasmid Mini Kit I.Secondly,to identify those plasmids with restriction endonuclease digestion and gel electrophoresis. Thirdly,pVAX1-SG,pVAX1-SSG and pVAX1 were purified and measured in large quantity with QIAGEN Plasmid Giga Kit,which were up to the requirements of the animal immunition,on the basis of correct identification.
Test two:To immunize BABL/c mice with the recombinant plasmids in order to observe the effect.Sixty-four 6-week-old male BALB/c mice were randomly divided into 8 groups of 8 mice.A:vaccinated with saline via intranasal route(negative control group); B:vaccinated with pVAX1 via intramuscular route(negative control group);C:vaccinated with pVAX1-SG via.subcutaneous injection in the submandibalar gland region route;D: vaccinated with pVAX1-SG via intramuscular route;E:vaccinated with pVAX1-SG via intranasal route;F:vaccinated with pVAX1-SSG via.subcutaneous injection in the submandibalar gland region route;G:vaccinated with pVAX1-SSG via intramuscular route H:vaccinated with pVAX1-SSG.via intranasal route;Every mouse was immuned a dose of 100μg in a week and for three times in all(saline group,100μl/only).The mice were weighted and killed after sampling.Then some important organs(heart,liver,spleen,lung, kidney) of the mice were separated for pathological examination.Using enzyme-linked immunosorbent assay(indirect ELISA) to test the level of specific antibodies S-IgA in saliva and IgG.in serum.
Test three:To immunize SD rats with the recombinant plasmids in order to observe the effect.First,to establish animal model of dental caries with SD rats.Then methods of division,the immunity means,immunity time and specific antibody detection are the same as the mice.To collect samples from first immune to age 80 days of mice,once a week.The classic of Caries were assessed according to Keyes scoring method.
Results:①pVAX1-SG,pVAX1-SSG and pVAX1 were isolated and purifiedy by large QIAGEN Plasmid Giga Kit in order to meet the requirements of the study;②The antibodies of S-IgA in saliva and IgG in serum were significantly increased and continued for several weeks after immunization in mice,at the same time nasal drip group and submandibalar gland group had induced higher than intramuscular group(P＜0.05).③The specific antibodies which pVAX1-SSG induced were more than pVAX1-SG induced under in the same immunition way,and with significant statistics(P＜0.05).④The level of caries are significantly lower in experimental groups than the negative control groups with significant statistics(P＜0.05).
Conclusions:①Gene vaccine pVAX1-SG and pVAX1-SSG can induce to improve markedly the level of specific in BABL/c mice and SD rats;②Gene vaccine pVAX1-SG and pVAX1-SSG can reduce caries by three different immune ways in SD rats.③nasal mucosal immunity may be the most effective immune way of gene vaccine pVAX1-SG and pVAX1-SSG.④pVAX1-SSG can be a better selection for specific antibodies than pVAX1-SG.
引文
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