Fusion Expression of apxIA Gene of Actinobacillus pleuropneumoniae in Escherichia coli and Its Product Purification

Abstract:

A 954 bp fragment corresponded to the main antigen determinant domain of apxIA gene of Actinobacillus pleuropneumoniae(APP)was amplified by polymerase chain reaction (PCR), from serovar I reference strain 259 and was ligated to pMD-18T vector for sequencing. After confirming the sequence correct, the extracted plasmid DNA was digested with EcoR I and Not I, followed by subsequent ligation into the prokaryotic expression vector, pGEX-4T-1, to construct an expression plasmid pGEX-4T-1/apxIA. The recombinant expression vector was transformed into E. coli DH5α for expression under the induction of 0.4 mmol/L IPTG. Glutathione Sepharose 4B was used for a further purification after the GST-fusion protein denatured and refolded by Urea. SDS-PAGE analysis revealed that the recombinant pGEX-4T-1/apxIA could be able to express efficiently in a form of inclusion body with 32% accumulated total amount of bacterial protein and the purity quotient of the GST-fusion protein was up to 95%. It is suitable for the clinical diagnosis and subunit vaccine of actinobacillus pleuropneumoniae infection.