[Construction of MyD88-Pseudomonas aeruginosa epitope DNA vaccine and its expression in eukaryotic cells].
OBJECTIVE
To construct the MyD88-Pseudomonas aeruginosa epitope vaccine and study its expression in eukaryotic cells.


METHODS
To design and synthesize an epigene containing three B cell epitopes of OprF and one foreign "promiscuous" T cell epitope by overlapping extension PCR. tPA signal encoding sequence was amplified by PCR and then it was inserted into the 5' terminus of the epigene to construct tPA-OprF. tPA-OprF and MyD88 were cloned into the expression vector pIRES and the recombinant plasmid pIRES-tPAOprF-MyD88 was constructed. The recombinant plasmid was transfected into COS-7 cells by electroporation. The expression protein of tPA-OprF and MyD88 was detected by Western blot.


RESULTS
The recombinant plasmid pIRES-tPA-OprF-MyD88 was successfully constructed. Western blot analysis indicated the tPA-OprF fusion protein was expressed in supertanant of COS-7 cells and MyD88 protein in COS-7 cells.


CONCLUSIONS
The recombinant plasmid pIRES-tPA-OprF-MyD88 has been successfully constructed and tPA-OprF and MyD88 protein can be highly expressed in transfected cells. It may be used as a potential candidate of preventive vaccine of pseudomonas aeruginosa.
Chen
Jian-guo
JG
People's Hospital of Jiangsu University, Zhenjiang, China. cjg02@126.com
Su
Zhao-liang
ZL
Liu
Ying-zhao
YZ
Wang
Sheng-jun
SJ
Liu
Xin-xiang
XX
Shao
Qi-xiang
QX
Xu
Hua-xi
HX
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D006801:Humans; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D054406:Pseudomonas Vaccines / Q000235:genetics / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D019444:Vaccines, DNA / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; C547605:OprF protein, Pseudomonas aeruginosa; D054406:Pseudomonas Vaccines; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA; D010959:Tissue Plasminogen Activator