Construction Of OmpA Deletion Mutant Of RA CH3 Strain And Study On The Biological Characteristics

Riemrella anatipestifer(RA)disease is a bacterial infectious disease.Currently,there are 21 identified RA serotypes,and the cross-protective immunity between serotypes is very poor.Because the conservative nature of RA ompA gene sequence is very high,many scholars are interesting in ompA gene,especially their immunological function,they want to solve RA different serotypes lacking of cross-type protective immunity by studying different serotypes ompA gene.In addition,the genomewide series of 5 strain of RA has been submitted to GenBank,the analytical results show that the RA coding protein has about 2000 pieces,but the biological function of protein A is unclear.OmpA protein,a major outer membrane protein,is the earliest identified RA and has immunogenicity.RA ompA gene among different serotypes has a high homology.Studying biological Characteristics of ompA gene will establish basis for preparing OmpA protein subunit vaccine preventing and curing RA disease.Further,as the main component of the outer membrane protein,the OmpA protein may also be involved in the transport of material,and maintaining the bacterial form and signal transduction.To analyze the effect of ompA genedeletion on the biological characteristics of RA,CH3ΔompA strain,an ompA gene deletion strain,was established using a suicide plasmid by homologous recombination.The left and right flanking sequences of ompA gene and a spectinomycin resistance cassette were amplified by PCR,respectively.These three fragments were ligated into the suicide plasmid vector pDS132 to produce a recombinant suicide plasmid pDS132-omp A-LSR.A bi-parental conjugation using E.coli S17-1(pDS132-ompA-LSR)as the donor strain and RA CH3 as the recipient strain was performed to transfer the plasmid pDS132-ompA-LSR to CH3.Transconjugants were selected on TSA agar in the presence of kanamycin and spectinomycin.The deletion of the ompA gene was confirmed by PCR and Western blot amplification,and the mutant strain was designated CH3ΔompA.In this study,the paper disk method was adopted to detect the susceptibility test in CH3,CH3ΔompA and CH3ΔompA(pRES0-ompA)strains,and analyze the drug resistance difference of CH3,CH3ΔompA and CH3ΔompA(pRES0-ompA).The results showed that,compared with deletion strain CH3ΔompA,wild strain CH3 had more sensitivity to β-lactam,chloramphenicol,tetracycline,doxycycline,erythromycin and other antibiotics.The drug resistance of complementary strain CH3Δomp A(pRES0-ompA)to tetracycline,doxycycline,erythromycin and chloramphenicol was between deletion strain CH3ΔompA and wild strain CH3.The reason that complementary strain CH3ΔompA(pRES0-ompA)had the enhancing resistance to penicillin antibiotics may be due to the shuttle expression plasmid pRES0 carrying ampicillin resistance bla gene in E.coli-RA.Wild strain CH3,deletion strain CH3ΔompA and complementary strain CH3ΔompA(pRES0-ompA)are tolerated to estomycin trimethoprim.The results showed that,compared with the wild-type strain CH3,the breeding speed of gene deletion strain CH3 ompA slowed down,the tolerance to NaCl(salt concentration)reduced,the sensitivity to some antibiotics increased,but the tolerance to SDS,EDTA,pH had no effect.The forming biofilm ability of gene deletion strain CH3ΔompA increased compared with wild strain CH3.