Prokaryotic Expression And Biological Characterization Of SLY And EF Of Streptococcus Suis Serotype2

Streptococcus suis serotype2(SS2) is an important worldwide zoonotic pathogen whichcauses arthritis, meningitis, pneumonia, septicemia, toxic shock syudrome and even death inhuman and swine. The outbreak and epidemic of SS2in1998and2005in our country causeda great number of pigs were infected and scores of people were killed. The pathogenesismechanism, detection methods, prophylaxis and treatment research of SS2had become ahotspot of the field of public health.In this study, SLY and EF genes were amplifid, cloned and prokaryotic expressed,,thensoluble rSLY and rEF were purified with a Ni-NTA affinity column. Hemolytic activity ofrSLY was determinated. The subunit vaccines of rSLY and rEF were prepared. IndirectELISA was established to detect the antibodies against rSLY and rEF. Finally the influence ofthe infection ability of Swine Influenza virus (SIV) was studied.1Prokaryotic Expression and Biological Activities Analysis of SLY and EF of StreptococcusSuis Serotype2SLY and EF genes were amplifid by PCR with genomic DNA of HA9801as templateand were cloned into prokaryotic expression vector pET-28a and transformed intoEscherichia coli for expression of rSLY and rEF. The cultured and induced E.coli cells werecollected after centrifugation and the bacterial pellet was lysed by ultrasonic treatment, andthen rSLY and rEF from bacterial cell were purified with a Ni-NTA affinity column.High-efficient expression and high-yield soluble rSLY with bio-activity was obtained. Thepurified rSLY presented a single band with the molecular weight of54ku.8mg of pure rSLYwas obtained from E.coli cells of100ml induced culture. rSLY had high hemolytic activityand its specific activity was4057HU/mg. It had hemolytic activity to many kinds of animals’erythrocytes, the erythrocytes of pig, rabbit, guinea pig were the most susceptible to it. rSLYmaintained stable hemolytic activity preserved at4℃for48hours. The optimal pH and ionicstrength condition for rSLY was pH6-7and500mmol/L, respectively. In this study, weobtained high-efficiently expressed soluble rSLY with bioactivity, and firstly reported effect of temperature, pH and ionic strength on the hemolytic activity of rSLY, these results will laya foundation for deeply research machinery of SLY and preparation of subunit vaccine anddiagnosis reagent for SS2.2Preparation and evaluation of subunit vaccines of SLY and EF of Streptococcus suis.New Zealand rabbit was used for study model. Subunit vaccines of rEF, rSLY, rEF+rSLYand inactivated oil-emulsion vaccines for SS2were prepared. The serum antibodygrowth-and-decline regulation against subunit vaccines was detected by ELISA. Rabbit wasinjected at two subcutaneous sites on back, the second immunity was carried out after10days,and serum wes separated at interval of10days, The results showed that there were strong andspecific antibodies acquired in blood-serum from rabbit after the subunit vaccination, and thenumber of antibody was in line with the growth law. After the first and second immunizationthe antibody titer in blood gradually raised until it approached the max, then remained highlevel for30d. It would be down gradually, and it remained a certain level at last. The wholecells SS2inactivated vaccine stimulates rabbit to produce the lower antibody titer of SLY andEF.3SLY and EF proteins of Streptococcus suis type2enhance the infection ability of the SwineInfluenza virus in MDCK cells.rSLY, rEF and mixtures of the two were effected on the MDCK cells that were added SIV.The slight CPE appeared that caused by SIV after adding MEM containing rSLY. After threetimes repeated freezing and thawing, the hemagglutination titer wes1:16, and it was muchhigher than blank control group. The results showed that rSLY enhance the infection ability ofthe Swine Influenza virus in MDCK cells.