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Objective To screen and prepare a broad-spectrum monoclonal antibody against Strep-tococcus pneumoniae ( S. pneumoniae) surface protein A ( PspA) and to evaluate its potential in clinical prac-tice. Methods Hybridoma cells were screened and inoculated into the abdominal cavities of BALB/c mice to prepare antibodies in ascites. Monoclonal antibodies were obtained by ammonium sulfate precipitation and protein A affinity chromatography and then identified by SDS-PAGE and Western blot. Their specificity, iso-forms and killing activities in vitro were analyzed. Results A broad-spectrum monoclonal antibody that rec-ognized PspA subclasses 2, 3 and 4 was obtained. Its in vitro killing rate against S. pneumoniae reached 40. 3％. Conclusions A broad-spectrum monoclonal antibody that could specifically bind to PspA was suc-cessfully prepared with a strong in vitro killing activity. This study provided reference for clinical diagnosis of S. pneumoniae-related diseases, quality assessment of S. pneumoniae vaccines and further research on mono-clonal antibody therapeutics.
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Objective To investigate the coverage of a recombinant protein vaccine based on pneumococcal surface protein A (PspA) from both family 1 and family 2.Methods One hundred and fifty-nine Streptococcus pneumoniae strains, including 47 invasive strains, were isolated from children in Nanjing Children′s Hospital.Cell lysates were prepared and reacted with three antibodies recognizing PspA -RX1, PspA-3296 and PspA-5668 for PspA typing by ELISA .Results Among 47 invasive isolates of 9 different serotypes, 10.7%were PspA family 1 and 89.3%were PspA family 2.Among all of 159 clinical isolates, 10.1% were identified as PspA family 1, 88.0%were family 2, while 1.9%of strains could not be typed by ELISA and PCR assays .None of strains belonged to PspA family 3.Conclusion The recombinant pro-tein vaccine based on PspA from both family 1 and family 2 has a broad coverage among clinical isolates and is potentially protective against both invasive and non-invasive pneumococcal diseases .
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Objective To compare the immunogenicity of pneumococcal surface adhesion A (PsaA) and pneumococcal surface protein A (PspA). Methods The variability of the genes and the expressed pneumococcal proteins PsaA and PspA was investigated by electrophoresis. Cross-reactivity of proteins with the antibodies induced by the corresponding proteins of Streptococcus pneumoniae serotype 5, 6B,1, 19F and 23F was researched by Western blot. The enzyme-linked immunosorbent assay (ELISA) was adopted to detect the antibody subclasses and the accessibility of antibodies induced by PsaA and PspA to the surface of the above intact strains. Cross-protection against challenging with Streptococcus pneumoniae strains was indagated in mice. Results Both proteins showed to induce the similar level of antibody subclasses.This study demonstrated that cross-reactivity of pneumococcal PspA was restricted in the same clade, which showed less extensive than pneumococcal protein PsaA. But antibody induced by pneumococcal protein PspA could be bound to the surface of the intact strains, which conduced the stronger cross-protection against inva sive strains. Conclusion The mice immunized with PspA protein cross-protected well against the invasive strains in which PspA belonged to the same clade 1 of family 1. It showed that pneumococcal protein PspA was more effective than PsaA in protection as composition of vaccine.
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The specific fragment of Pneumococcal surface protein A(PspA)and Pneumococcal Surface Adhesin A(PsaA)gene was amplified by PCR from Streptococcus pneumonia 5 and Streptococcus pneumonia 19.The amplified fragnent of PspA and PsaA gene was ligated into pET-27b(+)vector and transformed into BL 21 E.coli for expression and obtain the expressive production of PspA and PsaA.Induced by IPTG,the expression level was as high as 75 % of the total disolube protein.The result showed that the recombinant plasmid could express a specific 75 kDa and 37 kDa fusion protein in E.coli BL 21,which showed the good immunogenicity and a broadly cross reactivity with the other serotypes.
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Objective:To obtain purified PspA produced by prokaryotic expression system.Methods:Template DNA was isolated from cultured Streptococcus pneumonia TIGR4.By gene recombination technology in vitro,sequence encoding PspA antigen epitope was cloned into PET-32(a) expression vector.After being confirmed by sequencing,expressed antigen protein was identified by SDS-PAGE and Western blot.Results:The DNA sequence analysis confirmed that the cloned PspA gene was according to GenBank data.The PspA fusion protein was proved by Western blot.Conclusion:A highly expressed recombinant PspA protein was successfully obtained.
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Objective:To evaluate the ability of the cooperative protection of the Strep to coccus pneumoniae multivalent DNA vaccine containing lytA gene(N-acetylmuramoyl-L-alanine amidase,LytA)and pspA'gene(N fragment of pneumococcal surface protein A,PspA)against intraperitoneal Streptococcus pneumoniae D39 infection.Methods The genomic DNA of the standard Streptococcus pneumoniae strain D39 was isolated.LytA gene fragment was amplified by polymerase chain reaction(PCR)and cloned into the eukaryotic expression vector pcDNA3.1(+),which was transfected into BHK-21 cells using Lipofectamine TM2000,and then the expression of LytA protein was detected with Western-blot.Furthermore,three formats of recombinant plasmid DNA vaccines(pcDNA3.1-LytA+pcDNA3.1-PspA',pcDNA3.1-PspA',pcDNA3.1-LytA)were used to inject intramuscularly BALB/c mice,with pcDNA3.1(+)plasmid and PBS as controls.The levels of antibodies against PspA'and LytA proteins were checked.Live times of intraperitoneal infection mice by Streptococcus pneumoniae D39 in BALB/c models were analysis.Results:The three DNA vaccines all producted high levels serum special IgG antibodies.The anti-LytA antibodies level elicited by the DNA vaccine pcDNA3.1-LytA + pcDNA3.1-PspA'was higher than the DNA vaccine pcDNA3.1-LytA(P0.05).The result of the effect on intraperitoneal infection by Streptococcus pneumoniae D39 in BALB/c mice models showed the median live times in pcDNA3.1-LytA + pcDNA3.1-PspA'group was longer than pcDNA3.1-LytA group and the control groups(P0.05).Conclusion:The multivalent DNA vaccine of Streptococcus pneumoniae containing LytA gene and pspA gene had no the higher performance protection than the DNA vaccine only containing pspA gene against Streptococcus pneumoniae.It is should be further evaluated that LytA gene is concluded as the one of advantages candidate antigens of the Streptococcus pneumoniae multivalent DNA vaccine.
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To investigate the genetic variation within pspA from 17 clinical isolates of Streptococcus pneumoniae representing 12 capsular serotypes, we used specific PCR primers LSM12 and LSM2 derived from the DNA sequence of pspA of S. pneumoniae Rxl (type 2). We have found that all 17 isolates of S. pneumoniae have a pspA gene whose size ranges from 1.8 to 2.3 kb. RFLP analysis of the PCR-amplified pspA genes of the isolates exhibited distinct restriction patterns. Even within the same capsular type, the individual isolates of S. pneumoniae generally differed in PspA molecular masses and showed variabilities in the pspA gene locus. The nucleotide sequence of the pspA gene of S. pneumonaie KNIH1156 (type 19F) isolated from a blood specimen was determined. The sequence revealed an open reading frame of 1,827 bp nucleotides. Predicted size of the mature PspA was approximately 63 kDa. Deduced amino acid sequence of PspA of S. pneumonaie KNIH1156 revealed 57.0% identity with that of S. pneumonaie Rxl. Comparison of the nucleotide and amino acid sequences of PspA S. pneumoniae KNIH1156 (type 19F) with those of Rxl (type 2) showed considerable differences in the a-helical coiled-coil region of the two PspAs. These results suggest that the PspA of S. pneumoniae KNIH1156 has antigenic variations distinguished from those of Rxl strains.