Cloning And Expression And Immunodetection Of The Carboxy-Terminal Region Of The P1 Adhesin Protein Of Mycoplasma Pneumoniae

Mycoplasma pneumoniae is an important pathogen that leads to atypical pneumoniae and upper respiratory tract infections, such as bronchitis and pharyngitis. Post-infection, it is a common cause of myocardial damage, nervous system damage, kidney damage, infectious mononucleosis syndrome, Kawasaki disease and son on. Mycoplasma pneumoniae is consistent with the penicillin, and sensitive to macrolide, tetracycline. Therefore, it is important to fast diagnostic of Mycoplasma pneumoniae infection.Adherence to host respiratory epithelium is crucial in Mycoplasma pneumoniae infection and colonization. With multiple epitopes in Carboxy-Terminal Region of the P1, P1 is the major adhesion protein and immunogen of Mycoplasma pneumoniae.On basis of successful extract genomic DNA of Mycoplasma pneumoniae, the carboxyl-terminal gene of P1 was amplified by PCR with the template of genomic. Then P1 gene was cloned to the cloning vector pEASY-T1, and get recombinant plasmid pEASY-MP-P1F. Then pEASY-MP-P1F was sequenced. The correct P1F gene was cloned into pET32a(+) vector to construct the recombinant plasmid pET32a-MP-P1FE. After the expression vector pET32a-MP-P1FE was constructed, transformed it into E.coli BL21(DE3), and induced by IPTG. Recombinant protein was purified through nickel ion affinity chromatograph. SDS-PAGE showed that a recombinant protein (from 50 kDa to 60 kDa) was get. ELISA and Western Blotting were used to determine the antigenic of the recombinant protein. Purified recombinant protein immunized with patient's sera was found to be immunogenic. Part of the carboxy-terminal gene of P1 adhesin protein has been expressed and identified, which could be used as an antigen for immunodiagnosis and vaccine of Mycoplasma pneumoniae, as well as to understand the pathophysiology of the disease.