Prokaryotic Expression And Immunogenicity Analysis Of Mycoplasma Bovis LppB Protein

Cattles are the natural host of Mycoplasma bovis,and as of now there is no optimal solution for Mycoplasma bovis worldwide,Previously,antibiotic drugs such as macrolides were commonly used to treat mycoplasma diseases in cattle.and due to the misuse of antibiotics,Mycoplasma bovis is prone to drug resistance.The development of vaccines has become the focus of attention of scholars,and currently inactivated weak vaccines have been developed in the United States,however,the protection effect is not obvious.Therefore,there is an urgent need to explore more candidate antigenic targets for the development of safe and effective Mycoplasma bovis vaccines.In this study,the potential of LppB as an antigenic target was verified by cloning the Mycoplasma bovis LppB gene,prokaryotic expression of recombinant protein,polyclonal antibody preparation and reactogenicity analysis and rabbit in vivo protection assay.The main results are as follows.1.Cloning and bioinformatics analysis of LppB gene of Mycoplasma bovis.The LppB genes of three Mycoplasma bovis strains ZYJ5,GT01 and PG45 were cloned by designing specific primers and sent to the company for sequencing,and then compared for homology.The results showed that the LppB gene was highly conserved in Mycoplasma bovis strains,and the sequence homology of ZYJ5,GT01 and PG45clones was up to 100%with the popular Mycoplasma bovis strains HB0801,Tibet-10and Hubei-1 in China.Further analysis of its physicochemical properties showed that the LppB protein contained a lipoprotein signal peptide,the protein size was about 74k Da,the theoretical isoelectric point of the protein was 8.74,the overall mean value of hydrophilicity was-0.542,the predicted number of amino acids in the secondary structure of the protein was 619 AA,and the molecular weight was 69829.61 Da.2.Prokaryotic expression of Mycoplasma bovis LppB protein and preparation of polyclonal antibodies.The codons of Mycoplasma are used differently from those of E.coli,therefore,in this study,the termination codon TGA of Mycoplasma bovis on LppB gene expression was mutated to TGG and codon optimization was performed.The p ET30a-LppB plasmid was transformed into the expression bacterium E.coli BL21（DE3）and the LppB recombinant protein was obtained after 12h of IPTG（isopropylthiogalactoside）induced expression.Since the recombinant protein bears a His tag,the LppB recombinant protein was purified by affinity chromatography using Ni-NTA resin in this study,and validation by Western Blot showed that the target protein was obtained at a concentration of 1.6 mg/m L at 74k Da.The LppB polyclonal antibody was successfully prepared by emulsifying it with Fe-style adjuvant and immunizing Kunming mice.3.Determination of the potency of mouse polyclonal antibody to recombinant protein LppB and analysis of reactogenicity.The potency of the prepared LppB polyclonal antibody was determined by indirect ELISA tessellation titration at 1∶25600.The results showed that the LppB polyclonal antibody was able to recognize the corresponding position in the whole bacteriophage protein at 74 k Da effectively and singularly,proving its good reactogenicity.4.A recombinant protein LppB as a candidate anti-Mycoplasma bovis vaccine that contributes to immune protection in rabbits.Japanese Large-eared White rabbits were divided into immune attack group and attack group,and the test rabbits were immunized with LppB protein formulated with Freund’s adjuvant for 28 d.Mycoplasma bovis strain 07801（1×10⁸CFU/m L）was used for 5 d of continuous attack and observed for 2 d before sampling.The results showed that Mycoplasma bovis could be successfully isolated from nasal swabs and lung material.The body temperatures of the immune attack groups were all in the normal temperature range of 38.0-39.5℃,and the temperature of the attack group was unstable.The antibody level in rabbits showed an increasing trend,and the mean value of D_450nmwas 0.048 at the 0th d.After that,the antibody potency increased multiply at the 7^th,14^th,21^stand 28^thday.The highest mean value of antibody level in rabbits was 2.801 at the 35th d after the attack,which proved that the vaccine activated the immune response of the body and the antigen had good immunogenicity.The lungs of the attacked group were severely lesioned with parenchymal changes in some areas,and HE sections showed that the alveolar septum had lost its original compact structure and was infiltrated by a large number of inflammatory cells.In the immune attack group,the histopathological changes were relatively mild,and a small amount of inflammatory cell infiltration could be seen in the structural part of the alveoli in HE sections,which proved that the vaccine effectively protected the lungs of the rabbits and demonstrated the potential of LppB as an antigenic target.In summary,this study demonstrated that LppB protein has good immunogenicity through gene cloning,LppB protein expression,polyclonal antibody preparation and rabbit body test.The results of the study provide potential antigenic candidate targets for the development of Mycoplasma bovis vaccines.