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目的:将已构建的变形链球菌表面蛋白可变区重组质粒pEGFP-N1-Srv+经不同途径免疫SD大鼠,观察其在大鼠体内的原位表达,免疫定菌鼠后的特异性抗体形成及对龋齿的影响。方法:20只SD大鼠随机分为4组,重组质粒pEGFP-N1-Srv+经股四头肌肌肉注射和下颌下腺区皮下注射2种途径免疫大鼠,免疫组化观察重组质粒的原位表达;24只SD大鼠随机分为4组,免疫途径同上,免疫剂量为100μg/只,2周后加强免疫1次。应用间接酶联免疫吸附法检测血清特异性IgG抗体和唾液特异性sIgA抗体,Keyes龋齿计分法评估磨牙的患龋情况。采用SPSS17.0软件包对数据进行统计学分析。结果:①大鼠股四头肌细胞、下颌下腺组织内均见重组质粒的阳性表达。②重组质粒pEGFP-N1-Srv+经不同途径免疫定菌SD大鼠后,可检测到血清特异性IgG抗体和唾液特异性sIgA抗体水平升高,均于首次免疫后第4周达到高峰;该时段各组间的抗体水平,实验组均显著高于对照组(P<0.01);经下颌下腺区皮下注射后的sIgA抗体水平显著高于肌肉注射组(P<0.05)。③重组质粒免疫定菌鼠后,实验组和对照组间的龋齿计分无显著差异(P>0.05)。结论:重组质粒pEGFP-N1-Srv+能够在动物体内原位表达,并能诱导SD大鼠血清特异性IgG和唾液特异性sIgA抗体增高;经下颌下腺区皮下注射后的sIgA抗体水平显著高于肌肉注射,具有一定的免疫优势。但该区域单独的龋齿预防效果不明显。
OBJECTIVE: To construct the recombinant plasmid pEGFP-N1-Srv + of Streptococcus mutans and to immunize SD rats in different ways to observe its expression in situ in rats and the specific antibody after immunization And the impact of dental caries. Methods: Twenty SD rats were randomly divided into 4 groups. The recombinant plasmid pEGFP-N1-Srv + was immunized by intramuscular injection of quadriceps and subcutaneous injection of submandibular gland. The expression of recombinant plasmid was observed by immunohistochemistry 24 SD rats were randomly divided into 4 groups. The immunization route was the same as above, and the immunization dose was 100 μg / bird. Two weeks later, the animals were boosted once. Serum-specific IgG antibodies and salivary-specific sIgA antibodies were detected by indirect enzyme-linked immunosorbent assay (ELISA). Keyes caries score method was used to evaluate the caries status of molars. SPSS17.0 software package for statistical analysis of the data. Results: ① The positive expression of recombinant plasmid was found in rat quadriceps and submandibular gland. ② The level of serum-specific IgG antibody and salivary-specific sIgA antibody could be detected after the recombinant plasmid pEGFP-N1-Srv + was immunized by different pathways in SD rats, all at the 4th week after the first immunization. At this time The level of sIgA antibody in the submandibular gland subregion was significantly higher than that in the intramuscular injection group (P <0.05). ③ After immunization with recombinant plasmids, there was no significant difference in dental caries scores between the experimental group and the control group (P> 0.05). CONCLUSIONS: The recombinant plasmid pEGFP-N1-Srv + can express in situ in vivo in vivo and can induce the serum-specific IgG and salivary-specific sIgA antibody to increase in SD rats. The level of sIgA antibody in submandibular gland area after subcutaneous injection was significantly higher than that in muscle Injection, has a certain immune advantage. However, the effect of caries prevention alone in this area is not obvious.