口腔医学研究 ›› 2022, Vol. 38 ›› Issue (7): 640-646.DOI: 10.13701/j.cnki.kqyxyj.2022.07.012

• 儿童龋病研究 • 上一篇    下一篇

防龋基因疫苗pVAX1-GbpA/GBD缓释温敏型水凝胶不同途径免疫兔的效果研究

李虎1,2, 管晓燕2,3, 李敏3, 董竞男3, 肖茜文3, 白国辉2,3, 王铭蔚3, 刘建国2,3*   

  1. 1.遵义医科大学附属口腔医院儿童口腔科 贵州 遵义 563000;
    2.遵义医科大学 贵州 遵义 563000;
    3.贵州省普通高校口腔疾病研究特色重点实验室 贵州 遵义 563000
  • 收稿日期:2021-12-10 出版日期:2022-07-28 发布日期:2022-07-22
  • 通讯作者: * 刘建国,E-mail: liujg_001@163.com
  • 作者简介:李虎(1986~ ),男,贵州贵阳人,硕士,主治医师,主要从事龋病的病因及预防研究。
  • 基金资助:
    贵州省暨遵义市医用生物材料研发人才基地建设项目(编号:黔人领发[2018]3号;遵委[2019]69号);国家自然科学基金(编号:82160181);珠海市医学科研基金(编号:ZH3310200018PJL)

DNA Vaccine pVAX1-GbpA/GBD on Immunization of Rabbits by Different Methods

LI Hu1,2, GUAN Xiaoyan2,3, LI Min3, DONG Jingnan3, XIAO Qianwen3, BAI Guohui2,3, WANG Mingwei3, LIU Jianguo2,3*   

  1. 1. Department of Pediatric Dentistry,The Hospital of Stomatology,Zunyi Medical University, Zunyi 563000, China;
    2. Zunyi Medical University, Zunyi 563000, China;
    3. Special Key Laboratory of Oral Diseases Research, Higher Education Institution in Guizhou Province, Zunyi 563000, China
  • Received:2021-12-10 Online:2022-07-28 Published:2022-07-22

摘要: 目的:比较不同免疫途径下防龋疫苗pVAX1-GbpA/GBD聚乳酸乙醇酸-聚乙二醇-聚乳酸乙醇酸(PLGA-PEG-PLGA)水凝胶免疫兔后的免疫效应。方法:将重组质粒pVAX1-GbpA/GBD与空载体质粒pVAX1分别与PLGA-PEG-PLGA水凝胶震荡混匀。28只兔子随机分为7组:股四头肌注射疫苗组、口服疫苗组、鼻腔滴注疫苗组和颌下腺区皮下注射疫苗组、阴性对照组1股四头肌注射空载质粒pVAX1的水凝胶组、阴性对照组2口服空载质粒pVAX1的水凝胶组、空白对照组股四头肌注射生理盐水组。相同剂量进行3次免疫,第1次免疫1周后进行第2次免疫,2周后第3次免疫。采用酶联免疫吸附试验检测免疫前后唾液中和血清中的特异性IgA和IgG型抗GTF抗体水平。免疫组织化学法观察注射部位pVAX1-GbpA/GBD蛋白的阳性表达。结果:(1)防龋基因疫苗pVAX1-GbpA/GBD水凝胶经不同途径免疫兔后,免疫后2周4组防龋基因疫苗组的唾液中特异性IgA抗体和IgG抗体水平显著升高(P<0.05);IgA型抗GTF抗体水平在第6周达到最高,口服组在第4周IgA型抗GTF抗体水平高于其它6组;颌下腺注射组在第6、10周IgA型抗GTF抗体水平高于其它6组;口服组与颌下腺注射组在第8、12周IgA型抗GTF抗体水平高于其它组(P<0.05),但两者之间比较差异无统计学意义(P>0.05)。(2)免疫后2周4组防龋基因疫苗组的血清中特异性IgA抗体和IgG抗体水平显著升高(P<0.05);IgG型抗GTF抗体水平在第8周达到最高,口服组在第3、6、10周IgG型抗GTF抗体水平高于其它6组(P<0.05);颌下腺注射组在第4、8周IgG型抗GTF抗体水平高于其它6组(P<0.05)。(3)免疫组织化学染色结果显示4组防龋基因疫苗组的免疫部位可见pVAX1-GbpA/GBD目的蛋白的阳性表达,口服疫苗组的小肠黏膜部位的上皮细胞胞浆和颌下腺注射组的导管区域的细胞胞浆内中pVAX1-GbpA/GBD蛋白染色明显,表达强烈。结论:防龋基因疫苗pVAX1-GbpA/GBD水凝胶经4种途径免疫兔后均能诱导兔机体产生有效的免疫反应且维持较长时间,其中口服组与颌下腺注射组诱导的抗体水平更佳。

关键词: 葡聚糖结合蛋白, 基因疫苗, 水凝胶, 龋病, 免疫

Abstract: Objective: To compare the immune effects of poly (lactic/glycolic acid)-polyethylene glycol-poly (lactic/glycolic acid) hydrogel sustained-release anti-caries gene vaccine pVAX1-GbpA/GBD through different immunization routes. Methods: The temperature-sensitive biodegradable hydrogel PLGA-PEG-PLGA was mixed with recombinant plasmid pVAX1-GbpA/GBD and blank control. Twenty-eight New Zealand rabbits were randomly divided into seven groups (4 in each group). Plasmid pVAX1-GbpA/GBD was applied through intramuscular injection, oral administration, subcutaneous injection, or intranasal drip to rabbits. The control groups were intramuscular injection of pVAX1, oral administration of pVAX1 and hydrogel, and intramuscular injection of saline. All rabbits were immunized three times. After the first-time immunity, these rabbits need interval one week to strengthen the immunity by the same dosage, and then interval two weeks to strengthen the immunity 2nd time. The antibody responses induced by the vaccines were detected by indirect ELISA method. And the expression of recombinant protein was detected by immunohistochemistry technique. Results: (1) After 2 weeks of immunization, the levels of specific IgA antibody and IgG antibody in the saliva of 4 groups with anti caries gene vaccine increased significantly (P<0.05). The IgA anti GTF antibody level reached the highest in the 6th week, and the IgA anti GTF antibody level in the oral administration group was higher than those in the other 6 groups in the 4th week. The level of IgA anti GTF antibody in the submandibular gland injection group was higher than those in the other 6 groups at 6 and 10 weeks. The levels of IgA anti GTF antibody in the oral administration group and submandibular gland injection group were higher than those in the other groups at 8 and 12 weeks (P<0.05), however, no significant difference between them (P>0.05). (2) The levels of specific IgA antibody and IgG antibody in the serum of caries prevention gene vaccine group increased significantly 2 weeks after immunization (P<0.05). The level of IgG anti GTF antibody in the oral administration group was higher than those in the other 6 groups at 3, 6, and 10 weeks (P<0.05). The level of IgG anti GTF antibody in the submandibular gland injection group was higher than those in the other 6 groups at 4 and 8 weeks (P<0.05). (3) Immunohistochemical staining showed that the positive expression of pvax1 gbpA/GBD target protein was observed in the immune sites of four groups of anti caries gene vaccine group. The staining of pvax1 gbpA/GBD protein was obvious and strongly expressed in the cytoplasm of epithelial cells in the small intestinal mucosa of oral vaccine group and in the cytoplasm of ductal region of submandibular gland injection group. Conclusion: Through 4 immunization ways, the hydrogel sustained-release pVAX1-GbpA/GBD vaccine can induce effective systemic and mucosal immune response and maintain long time. The antibody levels induced by oral administration and submandibular gland injection are better.

Key words: glucan bind protein, gene vaccine, hydrogel, caries, immune