Sun Jing-hua, Niu Yu-mei, Fan Ming-wen, Xu Qing-an, Yang Xue-chao
Key Laboratory for Oral Biomedical Engineering of Ministry of Education, School of Stomatology, Wuhan University, China.
Zhonghua Yi Xue Za Zhi. 2009 Aug 25;89(32):2286-91.
To construct a new fusion anti-caries DNA vaccine pGJGAC/VAX encoding antigens of both S. mutans and S. sobrinus so as to enhance the protective effect of DNA vaccine against S. sobrinus infection.
The CAT fragment of S. sobrinus OMZ176 gtf-I was amplified by semi-nest PCR and then inserted into the plasmid pGJA-P/VAX to construct the recombinant plasmid pGJGAC/VAX. The CHO cell was transfected and the expression of fusion protein detected using cellular immunohistochemistry and Western blot. Mice were immunized with pGJGAC/VAX and control plasmids via the intramuscular (i.m) or intranasal (i.n) routes. During the experiment, blood and saliva samples were collected at a 2-week interval for antibody assay by ELISA. Rats were orally challenged with S. mutans Ingbritt or S. sobrinus 6715 and then immunized i.n with pGJGAC/VAX, pGJA-P/VAX or pVAX1. The Keyes method was used to determine the caries activity.
(1) CAT sequence was identical to the related sequence of gtf-I (OMZ176) in GenBank. The recombinant plasmid pGJGAC/VAX encoded the genes of antigens of both S. mutans and S. sobrinus. The expressed protein could respond to specific anti-PAc, anti-GLU and anti-CAT antibodies respectively. (2) As for antibody reactions, mice in the experiment group had significantly higher levels of anti-PAc, anti-GLU and anti-CAT IgG antibodies than those in the pVAX1 group (P < 0.01). The peak responses of specific anti-CAT antibodies were observed at 8 weeks (GAC/i.m) and 10 weeks (GAC/i.n) and were approximately 62.13 microg/ml and 11.43 microg/ml respectively. The peak responses of specific anti-CAT IgA antibodies were seen at 8 weeks (GAC/i.m) and 10 weeks (GAC/i.n) and were approximately 0.67% and 0.80% respectively. (3) In the group infected with S. mutans or S. sobrinus, the pGJGAC/VAX-immunized rats showed significantly fewer E, Ds and Dm lesions than pVAX1-immunized rats (P < 0.05) and decreased Ds and Dm levels than pGJA-P/VAX-immunized rats (P < 0.05) while there was no obvious difference in E lesions between the two groups (P > 0.05).
A new fusion anti-caries DNA vaccine pGJGAC/VAX encoding antigens of both S. mutans and S. sobrinus is constructed successfully and expressed correctly in eukaryotic cells. It induces effective mucosal and systematic humoral responses so as to provide a better protection against S. sobrinus.
构建一种新的融合抗龋DNA疫苗pGJGAC/VAX，其编码变形链球菌和远缘链球菌的抗原，以增强DNA疫苗对远缘链球菌感染的保护作用。
通过半巢式PCR扩增远缘链球菌OMZ176 gtf-I的CAT片段，然后将其插入质粒pGJA-P/VAX中构建重组质粒pGJGAC/VAX。转染CHO细胞，并用细胞免疫组织化学和蛋白质印迹法检测融合蛋白的表达。通过肌肉注射（i.m）或鼻内（i.n）途径用pGJGAC/VAX和对照质粒免疫小鼠。实验期间，每隔2周采集血液和唾液样本，用ELISA法检测抗体。用变形链球菌Ingbritt或远缘链球菌6715对大鼠进行口服攻击，然后用pGJGAC/VAX、pGJA-P/VAX或pVAX1进行鼻内免疫。采用凯斯法测定龋活性。
（1）CAT序列与GenBank中gtf-I（OMZ176）的相关序列一致。重组质粒pGJGAC/VAX编码变形链球菌和远缘链球菌的抗原基因。表达的蛋白可分别与特异性抗PAc、抗GLU和抗CAT抗体发生反应。（2）在抗体反应方面，实验组小鼠的抗PAc、抗GLU和抗CAT IgG抗体水平明显高于pVAX1组（P<0.01）。特异性抗CAT抗体的峰值反应在8周（GAC/i.m）和10周（GAC/i.n）时观察到，分别约为62.13μg/ml和11.43μg/ml。特异性抗CAT IgA抗体的峰值反应在8周（GAC/i.m）和10周（GAC/i.n）时出现，分别约为0.67%和0.80%。（3）在感染变形链球菌或远缘链球菌的组中，用pGJGAC/VAX免疫的大鼠的E、Ds和Dm损害明显少于用pVAX1免疫的大鼠（P<0.05），Ds和Dm水平低于用pGJA-P/VAX免疫的大鼠（P<0.05），而两组之间的E损害无明显差异（P>0.05）。
成功构建了一种新的融合抗龋DNA疫苗pGJGAC/VAX，其编码变形链球菌和远缘链球菌的抗原，并在真核细胞中正确表达。它诱导有效的黏膜和全身体液反应，从而对远缘链球菌提供更好的保护。
