融合防龋DNA疫苗pGLUA-P的构建及其细胞表达研究

目的 将变形链球菌（Streptococcus mutans)表面蛋白PAc编码A区和P区（A－P）的序列克隆到真核载体pGLU中，构建出GTF－PAc融合防龋DNA疫苗，并检测其在原核细胞和真核细胞中的表达。方法 将pCIA-P质粒中A－P片段序列与pGLU质粒连接，构建出GTF－PAc融合防龋DNA疫苗pGLUA-P。将pGLUA-P转化大肠杆菌BL21（DE3），以SDS－PAGE电泳鉴定目的蛋白GLUA－P的表达。通过脂质体将pGLUA-P转染大鼠原代肌母细胞，以免疫组织化学检测GLUA－P的表达。结果 GTF－PAc融合防龋DNA疫苗pGLUA-P经酶切分析证实携带GLU和A－P片段。pGLUA-P转化的大肠杆菌BL21（DE3）经IPTG诱导能够表达完整的融合蛋白。pGLUA-P转染的大鼠原代肌母细胞中可检测到融合蛋白的表达。结论 成功地构建了融合防龋DNA疫苗pGLUA-P，所携带的基因序列正确，能够在原核和真核细胞中表达正确的融合蛋白。

Construction and cellular expression of GTF-PAc fusion anti-caries DNA vaccine

Objective To construct a fusion anti caries DNA vaccine pGLUA P carrying GLU fragment from gtfB gene of Streptococcus mutans GS 5 and A P fragment including the A region and P region of PAc protein from a DNA anti caries vaccine pCIA P, and to investigate its expression in prokaryotic and eukaryotic cells. Methods The sequence of GLU fragment in pGLU plasmid was testified by DNA sequencing. The fusion anti caries DNA vaccine was constructed by ligating A P fragment from pCIA P to pGLU. The expression of GLUA P fusion protein in E. coli BL21 (DE3) was induced by IPTG and checked by SDS PAGE electrophoresis. pGLUA P was transfected in vitro to cultured rat primary muscle cells by cation liposome Dosper, and immunohistochemical method was used to test the expression of GLUA P fusion protein in cells. Results GLU sequence was identical with relative sequence of GTF I (GS 5 strain) in Gene Bank. Recombinant eukaryotic expression plasmid pGLUA P was confirmed to have both GLU and A P fragment. After pGLUA P was transferred into E. coli (DE3), it could express a new 115 000 protein by the induce of IPTG. Specific brown products could be found in the cytoplasm of cultured rat primary muscle cells transfected by pGLUA P. Conclusions Fusion anti caries DNA vaccine pGLUA P is successfully constructed and confirmed by sequencing and enzymes digestion. Fusion GLUA P protein can be correctly expressed in prokaryotic and eukaryotic cells.