| Streptococcus suis is an important swine pathogen that causes a number of pathological conditions, such as meningitis, endocarditis, arthritis, pneumonia and septicemia. There are currently 35 serotypes of Str. suis, among which type 2 is the most popular and pathogenic. Str. suis type 2 produce such virulence factors as suilysin, muraminidase-released protein(MRP), extracellular factor(EF), etc. Suilysin. belonging to thiol-activated cytolysin family(TACY), is a protective antigen capable of stimulating immune responses. Since suilysin from different serotypes of Str. suis is of high homogeneity, it might be able to provide cross protection against infections by different serotypes. Salmonella typhimurium, one of the invasive bacterial species, can be attenuated without loss of invasiveness. A number of attenuated S. typhimurium strains have been used as delivery vectors for foreign genes of bacterial or viral sources to the host tissues such as spleen and lymph node where they were expressed to induce specific immune responses. The main purposes of this study were to establish a PCR method for specific detection of Str. suis suilysin gene, construct the prokaryotic and eukaryotic expression vectors for suilysin gene and examine its expression using S. typhimurium as the host strain or delivery vector.From among 56 streptococcal isolates from diseased pigs, two from pigs with septicemia and meningitis were identified as Str. suis type 2 by the API kit and agglutination assay. These isolates demonstrated pathogenecity to rabbits and mice. The PCR primers based on the suilysin, MRP and EF genes of Str. suis type 2 could amplify the DNA fragments of expected length. A PCR assay for rapid and sensitive detection of Str. suis was then established. The PCR primers based on the suilysin gene could amplify a product of 1502bp length, which was digested by EcoRI into two fragments of 869bp and 633bp. With the nested PCR, a 1443bp fragment was produced. No PCR product was extended from Streptococcus equi subsp. zooepidemicus, Str. equinus, Str. acidominimus, Erysipelothrix rhusiopathiae and Actinobacillus suis. The PCR technique could detect as low as 100 bacterial cells. The results show that suilysin-based PCR could serve as a specific and sensitive diagnostic tool for detection of Str. suis.The suilysin gene of Str. suis type 2 isolate JX02 was amplified by PCR from bacterial genomic DNA, cloned into pMD18-T vector and then sequenced. The sequencing result showed that the PCR product was composed of 1494 nucleotides encoding a polypeptide of 497 amino acids. The first 27 amino acids were formed as the signal peptide. It is characterized by the conserve sequence (ECTGLAWEWWR) belonging to the TACY family. The amino acid sequence homology was above 99% with suilysins of Str. suis types 2, 1, 4, 8, 19, 23 and 28.Str. suis suilysin gene was cloned into a prokaryotic expression plasmid pBV220, and the recombinant plasmid was then transformed into the attenuated S. typhimurium strain ZJ111. Positive clones were screened by PCR and restriction enzyme digestion. Suilysin expression was analyzed by SDS-PAGE and western blot. PCR and restriction enzyme digestion confirmed the successful construction of the recombinant expression plasmid pBV220-SLY and its introduction into the attenuated S. typhimurium strain ZJ111. Suilysin was expressed in the recombinant strains and reactive to positive serum as shown by immuno-blotting.Suilysin gene of Str. suis strain JX02 was inserted into pcDNA3 under the control of human cytomegalovirus(hCMV) immediate early enhancer and promoter to construct recombinant eukaryotic expression plasmid pcDNA3-SLY. The pcDNA3-SLY was transformed by electroporation into the attenuated S. typhimurium strain ZJ111 resulting in ZJ11 l/pcDNA3-SLY, which was then used to infect the Vero cells. There were morphological changes of Vero cells transfected. DNA dot blotting revealed that the SLY gene was transferred into the Vero cells. A 58kD band of the suilysin was identified by SDS-PAGE.These re... |
PDF Full Text Request