Rana Aeromonas Hydrophila Aerolysin Gene Pronucleus Expression And Preliminary Analysis Of Immunogenicity

Wood frog "Red legs disease" is a major disease that harms Rana chensinensis raising industry, it brought badly economic loss to Wood frog's Mass breeding, characterized of fast infection and high mortality. Aeromonas hydrophila(AH) which attaches to serious aggressive to fish and batrachia plays a dominant role in the Red legs disease. Generally believed that this bacterial pathogenicity is closely related to its virulence factor, especially the Aer which is composed of 463 aminoacids and has a molecular weight of 51.5 ku, is one of AH's primary virulence factor possessing the peculiarities of hemolytic, cytotoxicity and bowel toxity. There are lots of serotypes of AH differing with the diversity of regions and hosts, which have limited the boundary of applying inactivated vaccine, cause of which calls for emergence of common protective antigen towards strains divided on serotypes and developing neotype subunit vaccine.We first designed a pair of orientiert primers targeted conservative regions of Aer gene according to the gene sequence announced on the GenBank, then apply PCR getting an object segment sized 1163 bp with the genome DNA of Aer from Rana chensinensis isolation as the template, after that, clone the segment to the vector pMD18-T to obtain a new recombinant, finally, we harvest a successful recombinant through sequencing and obtain 99% homology from BLAST analusis between the Aer gene of recombinant and DQ186611 published from the GenBank.We further cloned the Aer gene to the pronucleus expression vector pGEX-4T-1 and got an effective pronucleus expression vector pGEX-Aer through PCR and enzyme cutting verification. Afterwards, we focused on mass production and found the peak output of vector pGEX-Aer expressed in E.coli BL21 cost 8 hours inducing by IPTG(1 mmol/L).The fusion protein pGEX-Aer has a molecular weight of 69ku, most of which existed in the mode of inclusion body. Western blotting analysis suggests a good reactogenicity involving of the fusion protein. Then, purified the spot protein through affinity chromatography, immunized mice with it and tested the humoral and cellular immunity status applying Indirect ELISA and lymphopoiesis test. Results manifests that,organism unfolded preferable immunity (humoral and cellular immunity)level that stimulated by the protein which has indicated a good immunogenicity of this fusion protein and provided a desirable bedding to the future vaccine research on Rana chensinensis.