Cloning And Expression Of ApfA Of Actinobacillus Pleuropneumoniae And Its Immunogenicity

According to the sequence of apfA gene (GenBank accession number: AY235718) in GenBank, A pair of primers was designed to amplify apfA gene of SC-A strain by PCR with Primer 5.0 software. The product of PCR named SC-A-apfA is approximate497bp in length was amplified from the total DNA. The SC-A-apfA gene was cloned into the vector pMD18-T and the recombinant was named pMD18-T-SC-A-apfA. The gene includes a complete open reading frame which is 477bp in length and encoding a functional protein consists of 149 Aminos. Homology analyses indicated that the identity levels of deduced amino acid sequences of apfA among SC-A strain, 3 serotypes, 4 serotypes, 7 serotypes were also 99.3%.Singnal PV2.0 analysis showed that 1～33 amino acid residues of the peptide were signal pep- tide sequences. apfA deleted N-terminal signal peptide sequence was na- med MapfA. MapfA was inserted in BamHⅠand HindⅢmultiple cloning sites of the prokaryotic expression vector pET-32a(+) using the expression primers designed according to the sequence of apfA gene.pET-32a (+)-MapfA was identified and analyzed by corresponding restriction endonuclease on the basis of the genetic sites of pET32a and PCR, which was identified as the MapfA gene of SC-A strain.Purified pET-32a(+)-MapfA were transformed to E.coli BL21(DE3) and harvested Recombinant engineering strain.Identification with positive clone selection, SDS-PAGE analyses and Western blot analyses showed MapfA gene has been recombinated correctly with pET-32a(+) in BL21 (DE3) cell. The results affirmed the construction of Recombinant engineering strain of BL21(DE3) /pET32a-MapfA is successful. Purified pET-32a(+)-MapfA were transformed to E.coli BL21(DE3) and harvested Recombinant engineering strain. Identification with positive clone selection, SDS-PAGE analyses and Western blot analyses showed MapfA gene has been recombinated correctly with pET32a in BL21(DE3) cell. The results affirmed the construction of Recombinant engineering strain of BL21(DE3)/ pET-32a(+)-MapfA is successful.The Recombinant engineering strain of BL21(DE3)/ pET-32a(+)-MapfA was induced by using the EPTG and lactose as inducer. SDS-PAGE and Western blot analyses showed the MapfA gene has been highly expressed in E.coli BL21.The induced expression level between IPTG and lactose is undifferentiated. The molecular weight of Expressed fusion protein is about 32kDa. The content is approximately 42.5 percent in total protein of BL21. After the purified and annealed recombinated protein immuned the mice, the antibody titer elevated gradually along with the number of immuned mice using ELISA, and attacked the immuned mice with APP SC-A type by 5×LD50, the result of this assay certified that the recombinated protein was protective for the mice about percent 16.6.