中国生物工程杂志

Objective: Using the method of bioinformatics to predict the mutual epitope of Plasmid-mediated AmpC β-lactamases (pAmpCs) and to realize prokaryotic expression, purifica- tion and identification of AmpCs-Trx fusion protein. Method: Using the multi-sequence compa- rison tool to find various types of pAmpCs's relative conservative region, and using molecular biology software Biosun to predict the parameters of the accessibility, the antigenicity, the epitope, the flexibility and the hopp-woods, etc . Through comprehensive analyzing, find out the area of mutual epitope pAmpCs1. Based on the preference of the E. coli codon, obtain the nucleotide sequence pampcs1. Then the pampcs1 fragment was synthesized using overlapping PCR and the expression vector which is called pET32a (+) / pampcs1 was constructed. Adopting sequence verified, to make induced expression for the E.coli BL21(DE3) which contains the recombinnant plasmid. After the expressed product was analyzed by SDS-PAGE, to purify pAmpCs1-Trx fusion protein by using Ni-NTA affinity chromatography. And using the method of Western blotting to identify the antigenicity of pAmpCs1. Result: According to the result of prediction, the area of mutual epitope have been found out, and successfully established the recombinant expression vector pET32a(+)/pampcs1. BL21(DE3) strain was induced by IPTG, a specific expression band with a relative molecular mass 23×103 was detected by SDS-PAGE,and the purified fusion protein can be detected by multiclonal antibody against AmpC β-lactamases. Conclusion: Having been successfully established AmpCs1-Trx fusion protein prokaryotic expression vector and having been obtained the highly purified AmpCs1-Trx fusion protein. Initially prove the antigenicity of the AmpCs1-Trx fusion protein , settle a good foundation for preparing specific antibodies against pAmpCs1 and detecting pAmpCs.