Expression Of P116-N Terminal Specific Protein Of Mycoplasma Pneumoniae And Preparation Of Its Polyclonal Antibody

Objective: To study the preparation of one of the MP specific proteins of Mycoplasma pneumoniae(P116-609)and its polyclonal antibody.The collected serum samples were tested together with the prepared specific protein in conjunction with the P1protein-specific antigen previously expressed in the laboratory to analyze its specificity and sensitivity.Throat swab specimens were collected for culture,susceptibility analysis and Mycoplasma pneumoniae drug resistance analysis.Method: P116-609 protein expression and ELISA method to establish:-80 degree refrigerator Mycoplasma pneumoniae standard strain FH-MP for recovery.The culture was then performed using MP fast medium,and genomic DNA was extracted from the positive culture medium as a template.Reference Design Specific primers for PCR amplification of P1116-609 gene fragment.The obtained target gene fragment was purified and ligated into T vector.To be sent to the company after the test is correct,double digestion purification,and then connected to the p QE80-L vector.The constructed plasmids were transformed into DH5? competent E.coli and plated in 37°C incubator.Single colonies were picked into 5 ml LB medium,placed in 250 r/min shaking shaker shaking culture,centrifuged after 16 h Bacterial precipitation.DNA plasmid extraction kit,its plasmid extraction.The extracted plasmid concentration determination,and then double digestion.The digested solution was subjected to agarose electrophoresis and photographed under UV light.From the observation of the results of good or bad pick the appropriate bacterial liquid as a species of preservation.Expression of the target protein: The recovered strains were rescued for a small amount of expression,and the bacterial liquid before and after the induction was collected for SDS-PAGE electrophoresis to observe the expression of the bacterial target protein and to find out the most suitable conditions for protein expression.After confirming that the target protein of the strain is in line with the expected effect,carry out a large amount of bacteria shaking and centrifugation,and suspend with 100 ml of the prepared lysate B and add the protease inhibitor and the bacterial lysate.The whole process is operated on ice.After 20 min,sonication was performed: 300 Hz 5 s,5 s apart,total time 18 min.The broken liquid was centrifuged at 10000 r/min,and the supernatant was collected.Then add 50 ml lysis buffer B in the precipitate,after centrifugation in suspension,the supernatant was taken for later use.Before and after induction of bacterial liquid,supernatant of crude protein,inclusion body protein SDS-PAGE electrophoresis,and then staining with Coomassie Brilliant Blue 1 h,then decolorization decolorization 12 hours.Observe the expression of the target protein.Finally,the eluted solution with different p H values is eluted,the eluate is eluted,and then subjected to SDS-PAGE electrophoresis to stain the colloid to observe which gradient of the target protein is mainly in the eluate.Maintain the eluate containing the target protein,save for use.Further purify the target protein by NIE column and store for future use.Preparation of Polyclonal Antibody: New Zealand white rabbits were purchased from the Animal Experiment of Anhui Medical University and divided into four groups.New Zealand white rabbits were immunized with the target protein every week.The first two weeks of protein solution was mixed with complete Freund's adjuvant injection,the third four weeks of protein solution mixed with incomplete Freund's adjuvant injection of the ear vein.Finally,the venous blood was drawn from New Zealand White rabbits ear vein for Western-blot test to observe the effect of polyclonal antibody.ELISA method polyclonal antibody titer test to determine the potency of antibodies for subsequent test applications.Using the same method for laboratory preservation P1 protein strain recovery,the expression of the target protein.The serum-collected MP assay was then performed with the P116-609 protein itself expressed to compare the specificity and sensitivity of the single-specific protein assay and the combined assay.Result: The P116-609 gene plasmid was successfully constructed,and the protein expression and purification were well performed.The immunoreactivity of the expressed protein and the sera collected from MP-infected patients was verified by Western-blot.However,it is not immunoreactive with other sera from uninfected patients.The expressed P116-609 protein and P1 protein were antigen-coated in a96-well plate,and the commercial kit purchased was subjected to specificity and sensitivity analysis.Found that the combination of these two MP-specific protein detection effect is greatly improved.New Zealand white rabbits were immunized with the expressed protein,and the polyclonal antibody of this protein with higher titer was obtained.Conclusions: The experiment initially verified that P116-609 protein had good immunogenicity for detecting MP serum.At the same time combined with P1 protein coated 96-well plates,to improve the specificity and sensitivity of serum testing have a greater role.The higher titer of polyclonal antibody obtained from P116-609 protein immunized New Zealand white rabbits provided some theoretical help for the subsequent MP adhesion inhibition test.To some extent,it also shows that Mycoplasma pneumoniae some of its own specific antibodies produced in the body,Mycoplasma pneumoniae itself has a self-limiting role.