Shuttle vector construct and expression of IL1403-pHIV. A 900-bp fragment containing HIV env V2-V4 loop was fused with the surface-secreting signal and the anchor gene at the N- and C-terminus, respectively, to produce the shuttle vector (A); the HIV protein expression of the IL1403-pHIV was confirmed by Western blot analysis (B); and the expression on the surface of IL1403 was examined with flow cytometry analysis after surface staining with FITC-anti-Env Ab (C). The IL1403-pcyHIV–expressing HIV protein in cytoplasma was used as a negative control to rule out the penetration of the Ab. pAMb1 represents the replication region and the erythromycin resistance gene of pAMβ1 derived from Enterococcus faecalis. The replication region functions in L lactis, and the erythromycin resistance gene functions both in L lactis and in E coli. pUC19 represents the replication region of pUC19 functioning in E coli.