Expression Of Enhance Green Fluorescent Protein Was Used As A Species-Specific Marker In Streptococcus Suis Type 2 And Analysis Its Infectious Trend In Mice By Real Time PCR

SS2 is an important pathogen which has been associated with a wide variety of infections in swine, such as meningitis, septicemia, arthritis and pneumonia. And more disasters effect on the produce of swine in the world, especially in highly density. This organism has also been isolated from humans with meningitis or endocarditis and Toxic Shock Syndrome. Several putative virulence factors have been described, including the capsular poly-saccharide, muramidase released protein, extracellular protein factor, suilysin, encoding protein by orf2 , glyceraldehyde -3-pho -sphate dehydrogenase etc. But it is unclear that the SS2 how to breakthrough the barrier of mucosa and blood-brain. The SS2 recombinant with the Egfp reporting gene was constructed by homologous recombination, and then used the real-time PCR technology to reveal the SS2 infectious trend.1. Construction of pESEB vectorThe suylin gene(sly), enhance green fluorescence protein(Egfp)gene spectinomyc- in gene (spc) and bsly gene were integrated into pEVP3 in order to constructed the pESEB recombinant ,and then the recombinant was transformed into E.coil TOP-10.In the end , it was knew about by the PCR and endoenzyme digested that the four fragments have inserted into the predesigned site in pEVP3, And then extracted the recombinant of pESEB by TaKaLa Max-reagent Box to 1.5mg/Ml.2. Construction of the Egfp-HA9801 recombinantStreptococcus suis type 2 strain HA 9801 were grown in fresh THB (Todd Hewitt broth)culture with 40 mmol/L DL threonine to an OD₆₀₀ of about 0.4, Cells were then harvested by centrifugation and washed with ice-cold double-distilled water and ice-cold 0.3M sucrose. The cells were resuspended in 1 ml of 0.3M sucrose plus 15%(v/v) glycerol. Pulse was achieved with a setting of 25μF, 2.5kV and 230ft, After the electric pulse, the cells were diluted immediately in Todd-Hawit broth plus yeast and incubated for 2h 37℃, they were then plated on the plates containing spectinomycin. And then the Egfp-HA9801 recombinant was detected by the PCR and RT-PCR. After compared Egfp-HA 9801 recombinants with wild HA 9801 strains ,it has found that there are no change in the morpha structure, virulence, culture and biochemical characteristic.3. Analysis the SS2 infective trend in pigs by Real-time PCR.The taqman probe and primers was designed under the 142bp constant gene in egfp. The 142 bp genes fragment was integrated into pMD19-T vector, and then was transformed into E.coil TOP-10, and extracted in Mix-Reagent Box ,and used as the standard in the Real-time PCR. And was drawing the standard curve in order to set the best suitable parameters for the Real-time PCR. The Egfp-HA9801 recombinants were inoculated into the Balb/c mice by oral and intrapentoneal injection(i.p), and the control were inoculated with THB. After inoculated 8 hours, the mice were killed in each group every one hour. The bacterium cells were isolated from heart and blood were higher than the brain and liver in the i.p groups, however, in the oral groups ,the bacterium cells were isolated from brain and blood were higher than the liver and heart. It suggested that the SS2 infective trend was different in the two difference inoculate methods.