Prokaryotic Expression Of Outer Membrane Protein GcbG Of Avibacterium Paragallinarum And Construction Of Its Mutant With Capsule Transporter Gene HctC Deleted

Infectious coryza,caused by Avibacterium paragallinarum,is an acute respiratory disease of chickenswith cardinal symptom Head Tumidness. This disease causes growth impediment and raises eliminatingrate for chickens, which has led to a great loss for China Poultry Industry. Therefore research on thedevelopment of new-type genetic engineering vaccine which is safe, effective in producing immunereaction, is a main task at present.We cloned and sequenced the gcbG gene of Type B standard strain,which is495bp, constructedrecombinant pmkaryotic expression plasmid pGEX-5X-gcbG, transformed it into E. coli BL21(DE3),gotthe expression bacterial strain;Then we expressed the gcbG protein, optimized expression condition anddid immunogenic analysis, the results showed that we succeeded in expressing gcbG protein.,which is44ku and was mainly present in inclusion.Its optimum condition of the expression is IPTG1mM/L,temperature28℃, induction time5h. To purify recombinant protein and do westen-blot,the resultsshowed that the protein had specific reaction to positive Serumof Apg.we cloned and sequenced the HctC gene of Type B standard strain,which is1164bp.Then we choseposition cloning with Transposon, amplified the whole Hctc segment,connected with pGEM-Tplasmid,then we got the Gene-deleted segment by reverse PCR and reverse self-linkage,and thentransformed its into pemoC2suicide plasmid to constuct gene deleted transfer vector, which wastransferred to Avibacterium paragallinarum competent cell,then we got Avibacterium paragallinarum HctCgene-deleted strain by suicide plasmid-mediated homologous recombination. The results showed that thegene-deleted strain had no marked qualitative difference with wild strains in mycelial morphology,andgrowth rate was normal.