Cloning And Expression Of MRP Gene Of Streptococcus Suis Serotype 2

Streptococcus suis (S.suis) is an important swine pathogen that cause a number of pathological diseases, such as meningitis, endocarditis, arthritis and septicemia. There are 33 serotypes of S.suis, among which type 2 is the most popular and pathogenic. S.suis type 2 produce such virulence factors as muramidase-released protein (MRP), suilysin, extracellular factor (EF), etc. but MRP is one of the most important ones.The gene fragment of 34 to 813 bp (780bp) in length encoding the muramidase-released proteins was amplified from genomic DNA of Streptococcus suis type 2 strain QINGHAI by PCR. The PCR products was later cloned into T vector and transformed into host strain E.coli, and the plasmid DNA of positive clones was extracted, digested with enzymes and purified. Then the fragment was cloned into expression vector PET-28a(+). The recombinant plasmid was transformed into E.coli BL21. The fusion protein with a molecular weight of about 27KD could be expressed after induction with IPTG. and it possesses antigenic epitopes of muramidase-released protein.The gene encoding MRP of S.suis type 2 was cloned and subcloned into shuttle vector pAdTrack-CMV, forming transfer vector of pAdTrack-CMV/MRP, Linearized pAdTrack-CMV/MRP was electrotransformed into E. coli strain BJ5183 which is RecA+ and perform homologous recombination with supercoiled adenoviral vector pAdEasy-1 which contained in BJ5183 cells, the resultant recombinant which exposed inverted terminal repeat (ITR) sequence by restriction enzyme digestion was transfected into HEKAD-293 with lipoplast to produce the recombinant adenovirus (rAd-MRP). RT-PCR was used to detect the transcription of MRP, and serial passage and PCR were used to detect the stability of rAd-MRP. To evaluate the immune activity, rAd-MRP was inoculated to 28-day-old mouses by muscular injection and abdominal injection respectively, indirect enzyme linked immunosorbent assay (ELISA) was used to detect the titres of MRP specific antibody. the antibody titres reached 1:160.