Cloning, Expression Of Virulence-associated Genes Of Streptococcus Suis Serotype 2 And Their Application In Detection

Streptococcus suis serotype 2 is an important cause of meningitis, arthritis, endocarditis, septicemia, and sudden death in pigs. It is also increasingly becoming human health concern due to its zoonotic capabilities. Several outbreaks had occurred in China in recent past years. In 1998, the outbreak in Jiangsu killed 15 out of 29 human patients. In 2005, the other outbreak of human S. suis infection occurred in Sichuan, there were 204 people infected and 38 of them died. S. suis 2 infection could not only lead to enormously economic loss but also severely threat to human.To find out whether the outbreak in Sichuan was related to the mutations of virulence-associated genes of S. suis 2, sly, epf and mrp genes were respectively amplified from S. suis type 2 strain 449-1 isolated from Sichuan, cloned and sequenced. The nucleotide and amino acid sequences deduced from open reading frames( ORFs ) were analyzed. The sly gene of strain 449-1 was 1494 bp long encoding 497 amino acids. By comparion with S. suis strain 9801, p1/7, 1933 and SX332, the homology of nucleic acid was 100 %, 100 %, 99.7 % and 97.7 % respectively. The epf gene of strain 449-1 was 2499 bp long encoding 833 amino acids. By comparion with S. suis strain 9801, D282 and 396, the homology of nucleic acid was 99.9 %, 100% and 99.6 % respectively. The mrp gene of strain 449-1 was 3771 bp long encoding 125 amino acids. By comparion with S.suis strain 9801, D282 and 78, the homology of nucleic acid was 99.8 %, 100 % and 99.8 % respectively.The sao and cps2J genes of S. suis 2 were respectively cloned into prokaryotic expression vector pMAL-p2x and the recombinant plasmids( pMAL-p2x-Sao and pMAL-p2x-cps2J ) were constructed. An analysis revealed that amino acid sequence of sao gene of strain 449-1 was 270 bp less than the strain S735. The resulting recombinant plasmids were expressed in E. coli TB1 and induced by 1mM IPTG. Induction of recombinants harboring the malE-sao and malE-cps2J fusion genes led to the expression of an approximately 136 kDa MBP-Sao and 37.2 kDa MBP-cps2J fusion proteins. The amount of expressed proteins were mostly found in the cytoplasm of E. coli cells and respectively reached 12.3 % and 13.5 % of the total supernatant proteins. The fusion proteins were purified by amylose column. Both the MBP-Sao and the MBP-cps2J demonstrated specific reactivity to the positive sera collected from rabbits infected with S. suis 2. The results indicated that two kinds of recombinant proteins have high antigenicity.Rabbits were respectively inoculated intramuscularly with these two kinds of recombinant proteins. The results of specificity of antiserum detected by ELISA were similar to those deteced by coagglutination. The antiserum of fusion protein MBP-Sao reacted with Streptococcus suis serotype 2 and 9. The antiserum of fusion protein MBP-cps2J reacted with Streptococcus suis serotype 2, but not with Streptococcus suis serotype 9. This study suggested that these two kinds of fusion proteions could be used in the detection of S. suis.Rabbits were inoculated intramuscularly with these two kinds ofrecombinant proteins alone or in combination, respectively. These rabbits were challenged with 108 CFU/ml S. suis 2 strain 458. Results showed that fusion protein MBP-Sao group had the highest protection rate( 3/5 protected partly ), and other fusion protein inocoulated groups also had a low extent protection rate ( 2/5 protected partly, 2/5~3/5 protected partly ). The study would provide valuable support for further research of sao and cps2J genes of S. suis 2...