[Preparation of new protein carrier of vaccine against pneumococcal otitis media with genetic engineering technology].
OBJECTIVE
To prepare pneumolysin as a new protein carrier of vaccine against otitis media with genetic engineering technology and establish the base of the study on pneumococcal conjugative vaccines.


METHODS
Genomic DNA was isolated from streptococcus pneumoniae. A pair of primers which included two restriction sites was designed based on the published pneumolysin gene sequence. The pneumolysin gene was amplified from pneumococcal DNA with PCR technology. The restriction enzyme digested fragment was linked into the cloning vector PET-28a and the recombinant plasmid DNA containing pneumolysin was then transfected into host cell E. coli JM109 (DE3).


RESULTS
DNA fragments were subcloned to construct the complete pneumolysin gene by a conventional coning and PCR. The inserted pneumolysin gene sequence was confirmed by DNA sequencing and the pneumolysin protein was successfully expressed. The relative molecular mass of the expressed product was 52 000. The expressed product amounted to 8% of the total host cell protein.


CONCLUSIONS
The pneumolysin gene was successfully cloned into host cell using genetic engineering technology. The recombinant pneumolysin was expressed and purified for preparation. This work laid a foundation of the preparation of pneumococcal conjugative vaccines.
Chen
Bing
B
Department of Otology and Skull Base Surgery, Eye Ear Nose & Thorat Hospital of Fudan University, Shanghai 200031, China. bchen@citiz.net
Dai
Wen-jia
WJ
Wang
Zheng-min
ZM
Chen
Ze-yu
ZY
Chi
Fang-lu
FL
Li
Zhong-ming
ZM
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D005818:Genetic Engineering; D005822:Genetic Vectors; D010957:Plasmids; D022242:Pneumococcal Vaccines / Q000235:genetics; D013296:Streptococcus pneumoniae / Q000235:genetics*; D013301:Streptolysins / Q000096:biosynthesis* / Q000235:genetics
D001426:Bacterial Proteins; D022242:Pneumococcal Vaccines; D013301:Streptolysins; C032290:plY protein, Streptococcus pneumoniae