Cloning And Expression Of Fba Gene From Mycoplasma Hyopneumoniae、Analysis Of Immunogenicity And Activity

Fructose diphosphate aldolase (FBA) is a variety of vaccine and drug targets, Mycoplasma pneumoniae(Mycoplasma hyopneumoniae, Mhp) strong strain and the weak strain of FBA expression is different, so the research feature of pig Mycoplasma pneumoniae FBA immune to the disease prevention and control has an important significance.This test protein expressed in prokaryotic expression system of the Mhp fructose1,6-2-D-phosphate aldolase (FBA) protein, and application of the recombinant protein immune mice, using Mhp mycoprotein and FBA protein as envelope antigen in mice serum ELISA test, tested the FBA antigenicity.According to GenBank Mhp FBA gene sequences, primers were designed by the use of software Primer5.0. Gene sequencing was amplified by PCR,864bps fragment was found containing two rare codons. The front90bps was obtained by gene synthesis, the orther fragment was obtainde through the Splicing by overhang extension SOE-polymerase chain reaction (PCR) mutations, finally the mutated FBA gene was inserted into expression vector pET-28a(+),the recombinant expression plasmid pET-28a(+)/FBA was constructed.After it was successfully identifide,the fusion protein was inducede and expressed in E. coli BL21(DE3).We induced with1%IPTG after5h,the fusion protein expression of the highest.The fusion protein was purifide by Protino Ni-TED2000the Packed Columns. Western blot analysis showed that the fusion protein have satisfactory immunogenicity.In this study,we use recombinant protein FBA protein as antigen and coating antigen, by indirect ELISA method for detecting FBA protein immune antibody levels in BALB/c mice.Results show that the FBA protein can induce BALB/c mice to produce high levels of antibodies, has good immunogenicity.With different strains all mycoprotein as antigen, by indirect ELISA method for detecting FBA protein immune antibody levels in BALB/c mice.The results show that the level of the specific antibody in serum is higher.In purification of protein enzyme catalyzed reaction, enzymatic reaction, results show that the purified protein expression with aldolase activity.In this study, we successfully constructed the Mhp FBA recombinant bacteria, that express the Mhp recombinant protein FBA, this protein has the antigenicity and aldolase activity, thus to further study the Mhp pathogenic mechanism and establish a foundation for Mhp serological methods.