Site-directed mutagenesis of pneumolysin and series expression of pneumococcal surface protein A.

OBJECTIVE To amplify the mutated gene of Ply by using the site-directed mutagenesis techniques and construct the protokaryon expression vector containing pneumococcal surface protein A(PspA) and △ pneumolysin(Ply) genes by using molecular biology gene engineering methods.METHODS The primers of PspA and Ply were designed according to the gene sequence of Streptococcus pneumoniae TIGR4.The mutated Ply gene was obtained by the site-directed mutagenesis of Ply and PspA by PCR techniques.There fragments were linked to construct the prokaryotic expression vector pET32a-PspA-△Ply,which was identified by endonuclease digestion and DNA sequencing.The reassembled plasmid was then transferred into Escherichia coli BL21(DE3) and expressed through induced expression for different time,temperature and concentration,which was identified by Western blot.RESULTS Successful mutation of Ply mutant gene in △A146R147 base pair was confirmed by sequence.The result of DNA sequence analysis showed that the cloned PspA-△Ply gene sequence was completely corresponding to GenBank data.SDS-PAGE and Western blotting showed that the expressed PspA-△Ply fusion protein was about 1.20×105.CONCLUSION Successful site-directed mutagenesis of △Ply and construction of recombinant plasmids of pET32a-PspA-△Ply lay the foundation of the research on a new kind of anti-S.pneumoniae protein vaccine.