Preparation Of P116 Specific Protein Of Mycoplasma Pneumoniae And Study Of Polyclonal Antibody And Preliminary Study Of Mycoplasma Pneumoniae Mouse Model

Objective: To study the cloning and expression of the three-segment proteins FP2,FP3 and FP4 of the adhesion protein P116 gene specific to Mycoplasma Pneumoniae(MP),and the preparation of three corresponding polyclonal antibodies.Using the collected serum samples of positive patients to test its specificity and sensitivity.It provides a certain scientific basis for preventing and blocking the infection and transmission of Mycoplasma Pneumoniae.Methods: P116-FP2,P116-FP3,P116-FP4 protein expression and establishment of ELISA method: firstly,the FH-MP standard strain was resuscitated and cultured,and then its genomic DNA was extracted from the medium after a large amount of amplification and culture.The primers were designed by reference to common PCR.Amplify to obtain three target genes.Connect the target gene to the T vector,select the correct recombinant vector through blue and white spots,and then connect it to the p QE-80-L vector.Induced expression of the constructed three recombinant plasmids.SDS-PAGE electrophoresis to confirm protein expression and expand the cultivation of recombinant bacteria.Protein purification: firstly express the recombinant bacteria on a small scale,take the pre-induction bacteria liquid as a negative control,screen out the best conditions for protein expression,and express a large amount of protein under the most suitable conditions.Cleavage the expressed protein and add protease inhibitors.The expressed protein was sonicated under ice bath conditions.Centrifuge the sonicated bacterial solution to collect the supernatant,lyse the precipitate again,and take the supernatant after centrifugation.SDS-PAGE electrophoresis was performed on the bacterial liquid before induction,the bacterial liquid after induction,the crude protein of supernatant,and the inclusion body protein.Coomassie brilliant blue R-250 was used for staining.After the decolorization solution was decolorized,the protein situation was analyzed.The protein was eluted with different concentrations of imidazole eluent to determine the best elution gradient.Finally,it was eluted with a nickel column,and the purified protein was collected for use.Obtaining polyclonal antibodies: The experimental New Zealand white rabbits were grouped,and blood was taken as a negative control before immunization,and the rabbits were immunized with protein every week.In the first and second weeks the rabbits were injected with the protein suspension mixed with Freund’s complete adjuvant,and in the third and fourth weeks they were injected with the protein suspension mixed with Freund’s incomplete adjuvant.Blood was collected from rabbit ear veins for agar diffusion analysis to observe the effect of polyclonal antibodies.Finally,blood was taken from the rabbit to collect polyclonal antibodies.The immunogenicity of the obtained polyclonal antibody was detected by Western-blot with positive patient serum.The method of the BCA is used to detect the protein,and measure the concentration of the recombinant protein.The protein is used as the coating antigen.The checkerboard method from the rabbits is used to obtain the optimal conditions for the antigen-antibody reaction.Establishment of Mycoplasma Pneumoniae mouse model: c57 mice were divided into groups,and the mice were infected with the expanded cultured FH-MP standard strain bacterial solution once a day.Mice were repeatedly infected for three days.Alveolar lavage was performed on the fourth and seventh days,and lung tissues were taken.DNA extraction of alveolar lavage fluid,PCR identification.HE staining of lung tissue.Results: Three recombinant plasmid vectors of P116-FP2,P116-FP3,and P116-FP4 were successfully constructed.Western-blot experiment verified that it reacted with the corresponding antibodies in the serum of patients with Mycoplasma Pneumoniae positive,but could not react with the serum without MP infection.The checkerboard method is used to find the best antigen-antibody reaction system,which provides a theoretical basis for the subsequent development of ELISA kits.The lung tissue section of the mouse indicated that the mouse model of Mycoplasma Pneumoniae was successfully constructed,and the mouse alveolar lavage fluid also corroborated the establishment of the mouse model.Conclusion: It is preliminarily proved that the three proteins P116-FP2,P116-FP3,and P116-FP4 have immunoreactivity and the specificity of the Western-blot experiment as an antigen.Using it as a method for antigen detection of patient serum,the sensitivity of detection will be greatly improved.It provides certain help for clinical serological diagnosis of MP.The establishment of the mouse model of Mycoplasma Pneumoniae provides a theoretical basis for further prevention and treatment of Mycoplasma Pneumoniae.