[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.
Jia
Rong
R
Department of Endodontics, School of Stomatology, Wuhan University, Wuhan 430079, China.
Fan
Mingwen
M
Bian
Zhuan
Z
Guo
Jihua
J
Chen
Zhi
Z
Du
Minquan
M
D000818:Animals; D000831:Animals, Newborn; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003731:Dental Caries / Q000517:prevention & control; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism; D008562:Membrane Glycoproteins; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D022541:Streptococcal Vaccines / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use; D013295:Streptococcus mutans / Q000235:genetics / Q000276:immunology; D014162:Transfection; D019444:Vaccines, DNA / Q000235:genetics* / Q000627:therapeutic use
D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; C055764:S-layer proteins; D022541:Streptococcal Vaccines; D019444:Vaccines, DNA; D005964:Glucosyltransferases