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Cloning, expression and purification of truncated and hybrid fragments (families 1 and 2) OF PNEUMOCOCCAL SURFACE PROTEIN A (PSPA) AND PROTECTION AGAINST THE PATHOGENIC STRAIN 679/99 OF Streptococcus pneunomiae
Lopes LM; Bertoncini MDS; Miyaj E; Leite LCC; Cainelli Gebara VCB and Lopes APY
Centro de Biotecnologia – Instituto Butantan
Streptococcus pneunomiae is a pathogenic bacterium of great interest in public health since it causes diseases as pneumonia and meningitis. Currently vaccine consists of capsular polysaccharide of 23 different strains, which is not effective in children due to its thymus-independent immune response. PspA has been shown to be immunogenic and protective in experimental animals. It is a virulence factor that inhibits the activation of complement system avoiding fagocitosis. Based on its primary sequence PspA is divided in 3 families and 6 clades, which results in serological differences and consequently suggests the inclusion of PspAs of families 1 and 2 in order to have a broad protection spectrum. The present work describes the production of truncated and hybrid forms of PspA: PspA 1 (clade 1, family 1), PspA 3 (clade 3, family 2) and PspA 1-3 (clade 1 and 3), and the protection induced in immunized BALB/c mice against the pathogenic clade 3 strain of S. pneumoniae 679/99.
The PspA genes were isolated from genomic DNA of S. pneumoniae isolates. The hybrid was planned to contain the main immunogenic epitopes linked by the prolin rich region of the protein which acts in wild protein linking the colin tail (attached to cell surface) to the a-helix exposed region. The fragments of clades 1 and 3 were ligated using a Xba I site introduced by PCR in the pGEMT easy cloning vector and than linked in the expression vector pAE-6xHis between Xho I and Kpn I restriction sites. Proteins were expressed in E. coli BL21-DE3 and purification performed in an Akta Primme apparatus by Ni++ chelating chromatography. Recovery of the recombinant proteins was about 10-20 mg/L of culture. These proteins were able to induce the protection of mice against the challenge, with survival rates of 33% for PspA 1, 67% for PspA 1-3 and 83% for PspA 3. These are expected results showing a good protection for family 2 and a partial protection for family 1, and corroborate with our previous studies on serum cross reactivity among families.
Supported by: FAPESP
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