AIK is a novel cationic peptide with potential antitumor activity. In order to construct the AIK expression vector by Gateway technology, and establish an optimal expression and purification method for recombinant AIK, a set of primers containing AttB sites were designed and used to create the AttB-TEV-FLAG-AIR fusion gene by overlapping PCR. The resulting fusion gene was cloned into the donor vector pDONR223 by attB and attP mediated recombination (BP reaction), then, transferred into the destination vector pDESTl 5 by attL and attR mediated recombination (LR reaction). All the cloning was verified by both colony PCR and DNA sequencing. The BL21 F. coli transformed by the GST-AIR expression plasmid was used to express the GST-AIK fusion protein with IPTG induction and the induction conditions were optimized. GST-AIR fusion protein was purified by glutathione magnetic beads, followed by rTEV cleavage to remove GST tag and MTS assay to test the growth inhibition activity of the recombinant AIR on human leukemia HL-60 cells. We found that a high level of soluble expression of GST-AIK protein (more than 30% out of the total bacterial proteins) was achieved upon 0.1 mmol/L ITPG induction for 4 h at 37 °C in the transformed BL21 F. coli with starting OD₆₀₀ at 1.0. Through GST affinity purification and rTEV cleavage, the purity of the resulting recombinant AIK was greater than 95%. And the MTS assays on HL-60 cells confirmed that the recombinant AIK retains an antitumor activity at a level similar to the chemically synthesized AIK. Taken together, we have established a method for expression and purification of recombinant AIK with a potent activity against tumor cells, which will be beneficial for the large-scale production and application of recombinant AIK in the future.
Antimicrobial Cationic Peptides ; biosynthesis ; Antineoplastic Agents ; metabolism ; Escherichia coli ; metabolism ; Genetic Vectors ; HL-60 Cells ; Humans ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; Sequence Analysis, DNA

Objective To investigate the shedding of CAV2-ΔE3-CGS after immunization and the background of canine adenovirus (CAV) infection, and to establish a dual fluorescent quantitative PCR detection method for rabies virus (RV) and canine adenovirus type 2 (CAV2). Methods A dual fluorescent quantitative PCR detection method was established by designing specific primers and probes for E1 gene of CAV and G gene of RV for the detection of CAV2-ΔE3-CGS. Oral swabs, anal swabs and environmental samples of stray dogs from experimental animal farm and dog detention center were tested. Results The standard curves generated by this method were Y=-3.351 × logX + 44.895, R² = .999 and Y=-3.413 × logX + 45.192, R²=0.996, respectively. The linear relationships were good, and the minimum detection limits were both 102 copies/μL. CAV2-ΔE3-CGS was not detected in experimental animal farm. CAV was detected in dog detention center, and the positive rates were 5.88% (5/85) in oral swabs, 8.24% (7/85) in anal swabs, and 4% (1/25) in environmental samples. Conclusion The dual fluorescent quantitative PCR method can be used for the detection of CAV2-ΔE3-CGS after immunization and the investigation of CAV infection. The present study has shown that no CAV2-ΔE3-CGS has been detected after immunization and CAV infection rate of stay dogs is low in Shanghai. CAV2-ΔE3-CGS oral immunization meets requirement and is applicable.