Recombinant gene construction

A DNA fragment of 2L:EK : Exdn-4 consisting of 2 copies of GGGS as a linker, an enterokinase site, and Exendin-4 was chemically synthesized (Gene Universal, USA). 2L:EK : Exdn-4 was digested with XmaI and XhoI restriction endonucleases and ligated into the pTEX1::BiP : GB1:HL:GFP vector (Song et al., 2022), digested with the same endonucleases to give BiP : GB1:HL:2L:EK : Exdn-4. Subsequently, a DNA fragment, ddCBDm with 8xHis, that had been chemically synthesized (Gene Universal, USA) was digested with BamH1 and XmaI restriction endonucleases and ligated into pTEX1::BiP : GB1:HL:2L:EK : Exdn-4 digested with BamH1 and XmaI restriction endonucleases to give pTEX1::B:G:ddCBDm : Exdn-4 (BGC : Exdn-4). To add the ER retention motif HDEL at the C-terminus of BGC : Exdn-4, PCR was performed using primers XmaI-2L-EK-F and XhoI-HDEL-R ( Supplementary Table 1 ) and 2L:EK : Exdn-4 as template. The PCR product was digested with XmaI and XhoI restriction endonucleases and ligated to pTEX1::B:G:ddCBDm : Exdn-4 that had been digested with XmaI and XhoI restriction endonucleases to give pTEX1::B:G:ddCBDm : Exdn-4:HDEL (BGC : Exdn-4:HDEL).