Prokaryotic Expression And Immunoreactive Analysis Of The Annexin From Cysticercuscellulosae

Objective: To express AF239799-1 gene of Cysticercuscellulosae and analyze its immunoreactivity of expression products. Methods: The AF239799-1 gene from Cysticercuscellulosae was synthesized by the DNA automatic synthesizer in the way of the solid phase synthesis method of a phosphorous acid amide,it had two restriction enzymes sites both 5’NdeⅠ and 3’XhoⅠ. The gene was inserted into the prokaryotic expression vector p ET28a(+),then was transformed to Escherichia coli BL21(DE3). The extraction plasmid from Escherichia coli BL21(DE3) was identified by PCR and endonuclease digestion and gene sequencing,in order to determine the positive colonies. Then the positive colonies were induced with 1.0m M IPTG. Thereafter,expression products were analyzed by SDS-PAGE and purified by Ni-NTA affinity chromatography. In addition,the immunoreactivity of the purified recombinant proteins was analyzed by Western blot. Results: The length of synthetic AF239799-1 gene is 1 053 bp. The recombinant protein consist of 370 amino acids,which have a molecular-mass of 40.39 k Da and an isoelectric point of 6.80. PCR,double enzyme digestion and DNA sequencing all indicated that p ET28a(+)-AF239799-1 recombinant plasmid was constructed successfully, and successfully transformed into Escherichia coli BL21(DE3). Recombinant plasmid was expressed mainly in the form of inclusion bodies in the Escherichia coli BL21(DE3). Western blot showed that the recombinant protein could be recognized by serum of the pig infected taenia solium. Conclusion: The AF239799-1 gene of Cysticercuscellulosae can be highly expressed in prokaryotic expression system,and expression products have the immunoreactive characteristic.