Recombinant Anaerobic Bacillus Clostridium Egla P-her2/neu-il12 Fusion Gene Shuttle Expression Vector Construction

Objective: To construct a shuttle plasmid encoding a chimeric gene of Clostridium m saccharobutylicum eglA promoter and signal sequence (eglAp-extracell ular domain of human epidermal growth factor receptors 2 (hHer2/neu ECD)-human Interleukin-12 (rhIL12), pIMP1 eglA p-hHer2/neu ECD-rhIL12.Methods:The hHer2/neu ECD was amplified from pcDNA3.1 hHer2/neu by PCR. pcDNA6 hHer2/neu ECD-rhIL12 was prepared by inserting the hHer2/neu ECD fragment into the plasmid, pcDNA6 rhIL12. Template (55bp fragment of eglA p) was artificially synthesized and eglAp gene (335bp) was amplified by serial PCRs. Then T-eglA p was prepared. pcDNA6 eglA p-hHer2/neu ECD-rhIL12 was produced by inserting the eglA p gene into the plasmid pcDNA6 hHer2/neu ECD-rhIL12. Shuttle plasmid encoding a chimeric gene of Clostridium saccharobutylicum eglA promoter -Her2/neu ECD-IL12(pIMP1 eglA p-hHer2/neu ECD-rhIL12) was acquired by using in-fusion technique to insert the chimeric gene of eglA p-hHer2/neu ECD-rhIL12 into the pIMPl plasmid. Subsequently, the recombinant shuttle plasmid was identified by PCR amplifing, enzyme digesting, and sequencing.Results:All of the fragments, hHer2/neu ECD, eglAp and eglAp-Her2/neu ECD-rhIL12 were amplified, and the corresponding plasmids were prepared correctly. The shuttle plasmid of pIMPl eglAp-hHer2/neu ECD-rhIL12 was successfully constructed. No error was found both in the sequence and ORF of the acquired chimeric gene.Conclusions:A shuttle plasmid encoding a chimeric gene of Clostridium saccharobutylicum eglA p-hHer2/neu ECD-rhIL12 (pIMP1 eglA p-hHer2/neu ECD-rhIL12) was successfully prepared.