Establishment And Application Of Genetic Transformation Method Of Mycoplasma Gallisepticum

Mycoplasma gallisepticum(MG)is the main cause of chronic respiratory disease in chicken.The attenuated vaccine strain F36 is widely used tocontrol and prevent MG infection in China.The attenuated vaccine strain F36 is safe to chicken,has good ability of respiratory tract colonization,can induce mucosal immunity,and induce immune protection for a long time.In order to develop more potential use of F36 strain,,we proposed to construct gene operating system and explore F36 as vaccine carrier and lay foundation for multi-vaccine study.At present,we obtain the following results:1.Using the Lac Z gene of Escherichia coli as the reporter gene,combined with the Gap A promoter of MG S6 strain and the transposon p MT85,the genetic transformation method of Mycoplasma gallisepticum was established.The method is very easy to operate,without special equipment and supplies,and its conversion efficiency can meet the experimental requirements.p MT85 and recombinant plasmid were successfully transformed into MG F36 strain and MG S6 strain by the optimized PEG transformation method.As result,the transformed MG grew on FM-4 plate containing gentamicin and showed blue spot and fried egg liking colonies.After several transformations,a transposon library containing 600 mutants was obtained.Twelve colonies were randomly selected and passaged in FM-4 liquid culture and verified the stability by PCR.The results showed that the transformed MG was stable at least for 25 generations in vitro could amplify the gentamicin resistance gene on the transposon in 25 th passage.2.Based on the frame of plasmid p MD-18 T,fragment of 1800 b of MG replication region(Oric)and gentamicin resistance gene were cloned and inserted into p MD-18 T to construct the shuttle plasmid of E.coli-MG.And then the Lac Z gene expression cassette was also cloned into the shuttle plasmid,named as reporter plasmid to test the expression efficiency of shuttle plasmid.F36 strains were transformed with shuttle plasmids and reporter plasmids and plated on FM-4 solid medium containing gentamicin and X-gal.The plasmid-transformed F36 grown on the plate,and the colonies expressin Lac Z gene showed blue spots.But after passaged under no resistance conditions,some colonies from the blue spot clone lost blue spot,suggesting that there is a loss of plasmid in the cell.3.To further test the ability of the Gap A promoter,a recombinant transposon plasmid was construct.Based on the transposon vector p MT85,the plasmid was comprised of HA1 gene of avian influenza H5 subtype,which labeled with Flag at the 3 'end and fusioned with the Gap A promoter.After transformed into F36 and screened with gentamicin,two recombinant F36 strains were screenedand identified by PCR and Western blot.As results,HA1 protein was expressed in positive strains and reacted with AIV-H5 positive sera.Our results laid the foundation for the future development of the live vaccine of MG as multi-vaccine carrier.