中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (15): 2765-2768.doi: 10.3969/j.issn.1673-8225.2010.15.025

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

变异链球菌表面蛋白SpaP P区真核表达质粒的构建及表达

白国辉，刘建国，柴巧学，曲云鹏，管晓燕，韩  琪，杨德琴   

  1. 遵义医学院口腔医学院口腔内科学教研室，贵州省 遵义市  563000
  • 出版日期:2010-04-09 发布日期:2010-04-09
  • 通讯作者: 刘建国，博士，教授，主任医师，遵义医学院口腔医学院口腔内科学教研室，贵州省遵义市 563003 Liujg_001@163.com
  • 作者简介:白国辉★，男，1985年生，山西省侯马市人，汉族，遵义医学院口腔医学院在读硕士，主要从事口腔生物学的研究。 baiguohui1228@126.com
  • 基金资助:

    课题受贵州省优秀青年科技人才基金(黔科合人字[2005]0509号)，贵州省省长基金(黔科教办[2004]07号) ，贵州省教育厅重点项目(黔教科[2004]119号)资助。

Construction and expression of eukaryotic plasmid pVAX1-spap/P in mammalian cells

Bai Guo-hui, Liu Jian-guo, Chai Qiao-xue, Qu Yun-peng, Guan Xiao-yan, Han Qi, Yang De-qin   

  1. Department of Stomatology, School of Stomatology, Zunyi Medical College, Zunyi  563003, Guizhou Province, China
  • Online:2010-04-09 Published:2010-04-09
  • Contact: Liu Jian-guo, Doctor, Professor, Chief physician, Department of Stomatology, School of Stomatology, Zunyi Medical College, Zunyi 563003, Guizhou Province, China Liujg_001@163.com
  • About author:Bai Guo-hui★, Studying for master’s degree, Department of Stomatology, School of Stomatology, Zunyi Medical College, Zunyi 563003, Guizhou Province, China baiguohui1228@126.com
  • Supported by:

    Scientific and Technological Foundation for Excellent Youth of Guizhou Province, No. [2005]0509*; President  Foundation of Guizhou Province, No. [2004]07*; Educational Commission of Guizhou Province, No. [2004]119*

PDF

364

被引次数

12

摘要：

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-spap/P，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spap/P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。

关键词: 变异链球菌, 龋病, 疫苗, 表面蛋白, pVAX1, 真核表达

Abstract:

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 then analyzed 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 Ⅰrestriction 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.

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