Preliminary Establishment Of A Diagnostic Method For Pasteurella Multocida

Pasteurellosis is a common disease in animal husbandry.The pathogen is attached to healthy livestock and infects the body after a stress reaction,resulting in acute or chronic death,resulting in huge economic losses.The serotype of Pasteurella multocida is complicated,and the farmers lack animal protection awareness and scientific detection methods.Therefore,this experiment designed a rapid and efficient detection method based on the characteristics of the main toxin protein PMT.In the first experiment,the 3858 bp toxA gene sequence was analyzed by bioinformatics,and the highly conserved gene fragment(cstoxA)was obtained by BLAST method.The recombinant plasmid pMD-19T-cstoxA was constructed and sequenced to identify the correct recombinant plasmid pMD.-19T-cstoxA was named as the positive standard plasmid.According to the SYBR Green I fluorescence quantitative PCR reaction system designed by the laboratory,the experiment was optimized by primer concentration,primer amount,template amount,annealing temperature and reaction cycle number.The Ct value of the quantitative PCR reaction was calculated and the standard curve of SYBR Green I fluorescent quantitative PCR was determined.The standard curve was identified by sensitivity test,specific experiment and repetitive experiment.The results showed that the conservative analysis showed a highly conserved sequence of 104 bp,and the standard curve of SYBR Green I fluorescence quantitative PCR was constructed according to the optimized reaction system conditions.In the specific experiment,8 different strains were used.The strain was subjected to a qPCR reaction,and only a sample of Pasteurella multocida reached an effective response.In the sensitivity experiment,the qPCR reaction of different gradient samples showed that the sensitivity reached 102 copies/?L,which is much higher than the sensitivity of conventional PCR.In the repetitive reality,the standard deviation and the coefficient of variation are very low,which shows that the standard curve has good repeatability.In the second experiment,toxA-N and toxA-C were cloned and expressed by pET=28a prokaryotic expression system,and the expressed protein was purified by Ni-NTA gravity column.According to the spatial characteristics of PMT protein,the full-length 3858 bp toxA gene was expressed in sections,which were 1515 bp toxA-N gene and 2343 bp toxA-C gene.The recombinant plasmids pET-28a-toxA-N and pET-28a-toxA-C were constructed and transformed into BL21(DE3)competent cells for expression,and finally the expressed toxA-N and toxA-C proteins weresequentially subjected to SDS-PAGE,Western blot,protein solubility analysis experiments,and finally expressed toxA-N,toxA-C protein by Ni-NTA gravity purification.The results showed that the toxA-N and toxA-C genes of 1515 bp and 2343 bp were successfully amplified by PCR and expressed by the recombinant plasmid.The final recombinant plasmid pET-28a-toxA-N expressed about 60 ku.The inclusion body protein and the recombinant plasmid pET-28a-toxA-C expressed an inclusion body protein of about 90 ku in size.The protein eluate was analyzed to obtain a target band of the same size as the toxA-N and toxA-C proteins with fewer bands.In the third experiment,polyclonal antibody preparation and monoclonal antibody preparation were carried out by the principle of immune reaction.The prepared toxA-N and toxA-C protein immunogens were subcutaneously immunized in New Zealand white rabbits in stages,and rabbit blood was taken 45 days later to test the serum titer of the serum;the protein with better titer was taken for subsequent follow-up Preparation of monoclonal antibodies.The results showed that the antibody titer of toxA-N protein as immunogen reached 1:6400 in polyclonal antibody preparation,and the antibody titer of toxA-C protein as immunogen was 1:3200;toxA-N monoclonal antibody and toxA The titer of the-N polyclonal antibody is 100 times that of the latter.In this experiment,the SYBR Green I fluorescence quantitative PCR method for the identification of Pasteurella multocida was established by genetically modifying the solid-toxin protein(PMT)from the genetic level.Based on the principle of antigen-antibody binding,high-efficiency toxA-N polyclonal antibody and monoclonal antibody were prepared to construct an indirect ELISA diagnostic method for PMT protein.The combination of two diagnostic methods provides new ideas for the diagnosis of livestock diseases.