CAJ | 학술논문

背景:变异链球菌是龋病的主要致龋菌,针对介导变异链球菌非蔗糖依赖性黏附的重要毒力因子SpaP的基因疫苗在理论上能对抗变异链球菌黏附于牙面和进一步破坏牙体硬组织.目的:构建变异链球菌表面蛋白P区(SpaP/P)真核表达质粒pVAX1-spapiP,并观察其在哺乳动物细胞COS.7中的表达.方法:通过基因重组技术,构建真核表达质粒pVAX1-spap/P,并经酶切分析、测序分析鉴定正确后,采用脂质体转染法,将其转染至COS-7细胞中,然后经免疫组织化学SABC法检测其在细胞中的表达.结果与结论:真核表达质粒pVAX1-spap/P经EcoR Ⅰ和Xba Ⅰ双酶切分析,证实携带1.2 kb的目的基因spapiP片段,经测序分析,目的基因正向插入到预先设计的载体位点处.pVAX1-spap/P转染的细胞胞质呈褐色,pVAX1空载体质粒转染的细胞胞质中无着色.证实实验成功构建真核表达质粒pVAX1-spap/P,所携带的基因序列正确,能够在真核细胞COS-7中正确表达目的蛋白.
배경:변이련구균시우병적주요치우균,침대개도변이련구균비자당의뢰성점부적중요독력인자SpaP적기인역묘재이론상능대항변이련구균점부우아면화진일보파배아체경조직.목적:구건변이련구균표면단백P구(SpaP/P)진핵표체질립pVAX1-spapiP,병관찰기재포유동물세포COS.7중적표체.방법:통과기인중조기술,구건진핵표체질립pVAX1-spap/P,병경매절분석、측서분석감정정학후,채용지질체전염법,장기전염지COS-7세포중,연후경면역조직화학SABC법검측기재세포중적표체.결과여결론:진핵표체질립pVAX1-spap/P경EcoR Ⅰ화Xba Ⅰ쌍매절분석,증실휴대1.2 kb적목적기인spapiP편단,경측서분석,목적기인정향삽입도예선설계적재체위점처.pVAX1-spap/P전염적세포포질정갈색,pVAX1공재체질립전염적세포포질중무착색.증실실험성공구건진핵표체질립pVAX1-spap/P,소휴대적기인서렬정학,능구재진핵세포COS-7중정학표체목적단백.
BACKGROUND:Streptococcus mutans is the main pathogenic bacterium for dental caries. In terms of the gene vaccine's SpaP which mediate Streptococcus mutans non-sucrose dependent adherency, it antagonizes academically Streptococcus mutans, which adheres to dental facing and makes the tooth tissue worse. OBJECTIVE: To construct eukanyotic plasmid pVAX1-spap/P of surface protein antigen P of Streptococcus mutans and to evaluate the expression of the plasmid in mammalian cells COS-7. METHODS: The eukaryotic plasmid carrying encoding gene of spap/P of Streptococcus mutans was constructed and thenanalyzed by the restriction enzyme and sequence correctly, the plasmid was introduced into COS-7 cells by lipofectamine reagent. The transient expressed protein was detected by immunochemistry technique in COS-7 cells. RESULTS AND CONCLUSION: The expression of recombinant plasmid pVAX1-spap/P analyzed by the EcoR Ⅰ and Xba Ⅰ restriction enzyme confirmed carrying spap/P fragment of 1.2 kb. After sequencing, target gene was positively inserted into pre-designed point of the vector spaces. Positive expression was detected in plasma of the cells which were transfected with recombinant plasmid pVAX1 -spap/P. The cells which were transfected with pVAX1 were negative. The eukaryotic plasmid pVAX1 -spap/P was constructed successfully and target gene spap/P translated and expressed in COS-7 cells after transfecting with recombinant plasmid pVAX1-spap/P.