Construction Of Luciferase Tagged By Cell Penetrating Peptide And Its Application In The Detection Of In Vitro Tumor Chemosensitivity

ObjectiveAs the incidence of cancer continues to rise,malignant tumors have become common and frequently-occurring diseases that endanger human life and health,posing a serious threat to human survival.However,there are still obvious individual differences in the chemotherapy of clinical cancer patients.Different individual tumor tissues have different reactivity to the same chemotherapeutic drugs or chemotherapy regimens,even with the same pathological type and stage of the tumor patients with the same chemotherapy regimens are also some differences in sensitivity.So,the efficacy of tumor chemotherapy is still not ideal.Therefore,how to detect the chemosensitivity of tumor cells in patients before chemotherapy,screen the relatively effective anti-tumor drugs,avoid the blindness of chemotherapy and unnecessary adverse reactions,which become the key to effectively improve the efficacy of tumor chemotherapy.Analysis of intracellular adenosine triphosphate?ATP?can reveal how cells respond to different treatments,which is important for personalized medicine.In this study,we developed cell penetrating peptides?CPPs?tagged luciferase?TAT-LUC?for the in vitro chemosensitivity assay of tumor cells.Methods1.The plasmid containing luciferase was used as a template,the genes were obtained by PCR using primers?TAT-LUC?.And the prokaryotic expression vector pET28a-TAT-LUC was constructed.After transformed into BL21?DE3?,the target protein was induced by IPTG.2.The recombinant protein TAT-LUC was purified by nickel-ion affinity chromatography?Ni-NTA?.The activity of the recombinant protein was assessed by ATP standard solution and tumor cells.In addition,in the detection of intracellular ATP,kinetic studies were used to determine the optimal incubation time for TAT-LUC to enter the cell.The intracellular and extracellular ATP content was determined by luciferase?LUC?,which was not tagged with cell penetrating peptides;Meanwhile,the intracellular ATP content released by cell lysis was determined by the traditional luciferin-luciferase assay.The above two methods were compared to further assess the sensitivity and reliability of TAT-LUC.3.In vitro chemosensitivity of four tumor cells?Skov-3/DDP,A549/DDP,MDA-MB-231,Huh-7?was detected by TAT-LUC.At the same time,the TAT-LUC based method was compared with the MTT assay method to evaluate the performance of TAT-LUC method.Results1.The recombinant plasmid pET28a-TAT-LUC containing the cell penetrating peptides tagged luciferase gene was successfully constructed,and the target protein TAT-LUC was successfully expressed in BL21?DE3?.Besides,E.coli was cultured in LB medium containing Mg²⁺at concentration of 50 mM.When OD600reached 0.6,IPTG was added at a final concentration of 0.5 mM,and a large amount of highly active TAT-LUC was obtained after induction for 16 h at 22?.2.The expression vector pET28a for expression of TAT-LUC contains six histidine tags.Based on this characteristic,Ni-NTA column was used to purify the target protein TAT-LUC from recombinant E.coli after IPTG induction.For the free ATP assay,compared with LUC?R²=0.993?,TAT-LUC detected a relatively low luminescence intensity during the entire concentration range of ATP.The sensitivity of TAT-LUC was still capable of detecting 10 nM ATP,and activity of TAT-LUC was also proportional to the ATP concentration?R²=0.994?.For the intracellular ATP assay,the luminescence intensity measured with LUC treatment was significantly lower than TAT-LUC.TAT-LUC could enter the tumor cells after added to the medium for 2 min,and the activity was positively correlated with the cell number?R²=0.997?.In addition,ATP extracted from cells after lysing the cells by traditional luciferin-luciferase assay,which detected a minimum of 100 cells?R²=0.996?.And the traditional fluorescein-luciferase assay had a lower luminescence intensity over the entire cell range.In contrast,TAT-LUC was able to detect intracellular luminescence of at least 40 cells.The results showed that TAT-LUC was a reliable and sensitive tool for the measurement of ATP in living cells.3.TAT-LUC successfully detected the chemotherapeutic sensitivity of four tumor cells?Skov-3/DDP,A549/DDP,MDA-MB-231 and Huh-7?.The reliability of the TAT-LUC based method was proved by comparing with the results of chemosensitivity test by MTT assay method.ConclusionWe created a fusion protein successfully,cell penetrating peptides tagged luciferase TAT-LUC.Compared with LUC,TAT-LUC was capable of penetrating the cells.The TAT-LUC based assay had a good sensitivity,which could detect at least 10 nM ATP or 40 tumor cells.The TAT-LUC based assay further analyzed the chemosensitivity of four tumor cells?Skov-3/DDP,A549/DDP,MDA-MB-231,Huh-7?and demonstrated its reliability and sensitivity for in vitro tumor chemosensitivity.The greatest advantage of TAT-LUC based assay is the ability to measure ATP levels in living cells quickly and directly,without the need to lyse the cells.Which avoids the ATP degradation and loss due to cell lysis.Single or few tumor specimens can also be used for drug sensitivity testing,improve the detection speed greatly.In addition,it enables real-time reflection of tumor drug resistance and accelerates the detection rate,which can be a valuable aid for personalized cancer chemotherapy.