The Proteins Analysis Of Mycoplasma Bovis From Different Generations And Evaluation Of The Immune Effects

Mycoplasma bovis is an important pathogen,that can cause many diseases leading to huge economic losses all over the world.Epidemiological surveys show that the infection rate of M.bovis is increasing year by year.With the extensive use of antibiotics in clinical treatment,M.bovis strains gradually have resistant to drugs,so that vaccine is an important measure to prevent and treat related diseases caused by M.bovis.The researchers hope to increase the number of live bacteria per unit volume of M.bovis isolates in a short period of time by changing the composition of the medium,thereby reducing the production cost of the vaccine,but the results are not satisfactory.The study found that the number of viable cells per unit volume of culture medium of M.bovis can be changed in magnitude by serial passage in vitro.How does the expression level of M.bovis protein change during this process?If some important protective antigens are lost,it will lead to a decline in immune protection.In this study,iTRAQ,Western blot and qPCR were used to analyze the total protein expression levels of different generations of M.bovis,in order to screen the generation that the expression levels of important immunoprotective antigens have Minor change,while the amount of bacteria have significant growth.The best generation strains were prepared as inactivated vaccines,and preliminary clinical trials were conducted.The main conclusions are as follows:1.In this study,an epidemiological survey of M.bovis infections in a large-scale cattle farm in the surrounding area of Hohhot was carried out for 3 years.It was found that the seroprevalence of M.bovis in recent years has increased year by year,and the infection rate is as high as 90%.Through the evolutionary analysis of multiple strains by the sequence of 16S rDNA,it was found that they have little difference in evolution distance laboratory between our laboratory isolates strains and the representative regions of other regions included in the database.The domestic strain of M.bovis relatively conservative.2.The growth curve of 7 generations of NM-28 strain was determined by CFU.With the increase of the generation,the number of viable cells per unit volume increased,and the time required to enter the plateau period was also shortened.The number of live bacteria in the 120th generation and later generations can reach 1011 cfu/mL,and it enters the plateau period at 24 hours.The other 4 lower generations enter the plateau period for 30 hours,and the highest number of live bacteria only reaches 1010 cfu/mL.The M.bovis NM-28 isolate can be continuously passaged in vitro to increase the number of viable cells per unit volume and increase by an order of magnitude.3.Proteomics was used to study the differential protein expression between NM-28-F3 and NM-28-F60 by iTRAQ.According to bioinformatics analysis,differential proteins are mainly involved in ribosome composition or protein synthesis.Membrane proteins with immunity,was no difference in expression levels between NM-28-F3 and NM-28-F60.4.According to the results of proteomics,P81,P48,EF-Tu,2 membrane proteins and 1 lipoprotein were screened,expressed by prokaryotic,and New Zealand rabbits were immunized to obtain high serum-free serum with titer greater than 1:1600.Through Western blot analysis,confirmed the reactivity of recombinant protein.5.The expression levels of 15 protein mRNAs in different generations were studied by real-time PCR.The results showed that the NM-28-F120 and NM-28-F3 had the similar expression levels.Western blot was used to study the expression levels of these six proteins in different generations of M.bovis NM-28 strain,and all of them were expressed and the expression level was not obvious.Combined with the growth curve measurement results,the NM-28-F120 and above generations were an ideal strain for the inactivated vaccine of M.bovis,which could reduce the production cost of the inactivated vaccine while ensuring the immune effect.6.Through clinical trials,M.bovis NM-28-F120 and NN-28-F150 inactivated vaccine can induce the body to produce an immune response,and can also immunologically infect yak,reducing the incidence and prevalence of infection.There is no statistically significant difference between them.Taking into account factors such as cost,NM-28-F120 is the best generation for vaccine strains.