Construction and expression of circular RNA in vitro / 中国生物制品学杂志

@#Objective To construct encoding RNA that can be cyclized in vitro by using the permuted intron exon(PIE)strategy in the maturation process of eukaryotic mRNA,and transfect it into HEK-293T cells for expression.Methods The sequences of 5'and 3'cyclic arms with groupⅠcatalytic intron,the internal ribosome entry sites(IRES)of Coxsackievirus B3(CVB3)and the target gene were selected to construct the template plasmid. Linearization plasmid template obtained by PCR was used to synthesize linear RNA through in vitro transcription(IVT),which then started in vitro cyclization(IVC)by the addition of cyclization reagents to obtain circular RNA(circRNA). RNA cyclization was confirmed by agarose gel electrophoresis and ribonuclease R(RNase R)digestion. HEK-293T cells were transfected with circRNAs respectively carrying enhanced green fluorescent protein(EGFP),firefly luciferase(Fluc),and influenza virus hemagglutinin(HA)IVR-180 genes,to verify their expression with in vitro.Results With RNA cyclization,the main band of agarose gel electrophoresis became smaller and small fragments appeared. After RNase R digestion,only some circRNA bands remained.HEK-293T cells transfected with EGFP-circRNA showed significant green fluorescence under the fluorescence microscope.The Fluc expression values of HEK-293T cells transfected with Fluc-circRNA were on average 20 times higher than non cyclized RNA,and the relative light unit(RLU)scaled up with the increase of Fluc-circRNA transfection dose. Western blot analysis showed that HA protein was successfully expressed in HEK-293T cells transfected with HA-circRNA.Conclusion In this study,linear RNA was successfully cyclized in vitro and different proteins were expressed,which lays a foundation of the research of new influenza vaccines and mRNA vaccines.

Validation and invalidation of SARS-CoV-2 main protease inhibitors using the Flip-GFP and Protease-Glo luciferase assays

SARS-CoV-2 main protease (Mpro) is one of the most extensively exploited drug targets for COVID-19. Structurally disparate compounds have been reported as Mpro inhibitors, raising the question of their target specificity. To elucidate the target specificity and the cellular target engagement of the claimed Mpro inhibitors, we systematically characterize their mechanism of action using the cell-free FRET assay, the thermal shift-binding assay, the cell lysate Protease-Glo luciferase assay, and the cell-based FlipGFP assay. Collectively, our results have shown that majority of the Mpro inhibitors identified from drug repurposing including ebselen, carmofur, disulfiram, and shikonin are promiscuous cysteine inhibitors that are not specific to Mpro, while chloroquine, oxytetracycline, montelukast, candesartan, and dipyridamole do not inhibit Mpro in any of the assays tested. Overall, our study highlights the need of stringent hit validation at the early stage of drug discovery.

Rapid establishment of a mouse model of MERS-CoV pseudovirus infection based on DPP4 transduction / 中华微生物学和免疫学杂志

Objective To rapidly establish a mouse model for optical imaging of the dynamical process of pseudotyped Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Methods In vitro expression in HeLa cells and functions of hDPP4 encoded by recombinant adenovirus Ad5 and pseudo-typed MERS-CoV were verified. The recombinant adenovirus expressing hDPP4 (Ad5-hDPP4) was injected into BALB/ c mice, which were then injected with pseudotyped MERS-CoV expressing firefly luciferase at a titer of 3×107 TCID50(50％ tissue culture infective dose) via intrathoracic (I. T. ) or intraperitoneal (I. P. ) injection. MERS-CoV infection and tissue distribution were observed using optical imaging techniques. Re-sults hDPP4 and firefly luciferase were efficiently expressed in HeLa cells. In BALB/ c mice injected with Ad5-hDPP4 via I. P. , firefly luciferase expression were detected in abdomen between 48-96 h after pseudo-typed MERS-CoV infection. The expression of firely luciferase was also detected in chests of BALB/ c mice injected with Ad5-hDPP4 via I. T. around 48 h after pseudotyped MERS-CoV infection. Conclusions This study reported a simple and rapid method for establishing a mouse model for conveniently and dynamically monitoring pseudotyped MERS-CoV infection, which might provide an effective means for in vivo evaluation of neutralizing antibodies or entry inhibitors by visualization with optical imaging techniques.

Construction of lung tumor cells using RFP and Fluc dual-labeled system / 国际生物医学工程杂志

Objective To establish a human lung cancer cell line that can stably express firefly luciferase (Fluc) and red fluorescent protein (RFP) gene so as to lay the foundation for the further establishment of a live-imaging lung cancer xenograft model in nude mice and therapeutic research.Methods The lentiviral vector pHBLV-FlucRFP containing luciferase and red fluorescent protein was constructed and then transfected into 293T cells for virus packaging.The complete virus was used to infect human lung cancer cell lines A549,H1975 and human B-cell lymphoma cell line K562.The stable cell lines were obtained by puromycin selection.Fluorescence microscopy and quantitative PCR were used to confirm the RFP and Fluc expression.Results The lentiviral vector pHBLV-FlucRFP was successfully constructed.Cancer cell line A549,H1975 and K562 stably expressing Fluc and RFP was obtained.The real-time quantitative PCR results showed that the relative expression of Fluc gene in the three stable infected cells was much higher than that in the corresponding wild-type cells,and the differences were statistically significant(all P＜0.05).Conclusion The human lung cancer cell line A549,H1975 and human B-cell lymphoma cell line K562 with dual expression of RFP and Fluc were obtained,which provided a new model of fluorescent cells for in vivo imaging of immunodeficient mouse models such as nude mice.

A New Indicator Cell Line Established to Monitor Bovine Foamy Virus Infection / 中国病毒学·英文版

In order to improve the accuracy for quantitating the bovine foamy virus(BFV)in vitro,we developed a baby hamster kidney cell(BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter(LTR,from -7 to 1012). The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR. One clone,designated BFVL,was selected from ten neomycin-resistant clones.BFVL showed a specific and inducible dose-and time-dependent luciferase activity in response to BFV infection.Although the changes in luciferase activity of BFVL peaked at 84 h post infection,it was possible to differentiate infected and uninfected cells at 48 h post infection. A linear relationship was established between the multiplicity of infection(MOI)of BFV and the activated ratio of luciferase expression in BFVL. Moreover,the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection. These findings suggest that the BFVL-based assay is rapid,easy,sensitive,quantitative and specific for detection of BFV infection.

Intravenous KITENIN shRNA Injection Suppresses Hepatic Metastasis and Recurrence of Colon Cancer in an Orthotopic Mouse Model

KITENIN (KAI1 C-terminal interacting tetraspanin) promotes invasion and metastasis in mouse colon cancer models. In the present study, we evaluated the effects of KITENIN knockdown by intravenous administration of short hairpin RNAs (shRNAs) in an orthotopic mouse colon cancer model, simulating a primary or adjuvant treatment setting. We established orthotopic models for colon cancer using BALB/c mice and firefly luciferase-expressing CT-26 (CT26/Fluc) cells. Tumor progression and response to therapy were monitored by bioluminescence imaging (BLI). In the primary therapy model, treatment with KITENIN shRNA substantially delayed tumor growth (P = 0.028) and reduced the incidence of hepatic metastasis (P = 0.046). In the adjuvant therapy model, KITENIN shRNA significantly reduced the extent of tumor recurrence (P = 0.044). Mice treated with KITENIN shRNA showed a better survival tendency than the control mice (P = 0.074). Our results suggest that shRNA targeting KITENIN has the potential to be an effective tool for the treatment of colon cancer in both adjuvant and metastatic setting.

Establishment of reporter gene labeled HCV full-length genome replication cell model / 中华微生物学和免疫学杂志

Objective To establish a stable HCV full-length genome replication cell model which is labeled with reporter gene and easyly to quantify intracellular HCV proteins and RNA level. Methodsneo gene was inserted into Luc-JC1 to make Luc-JC construct. Luc-JC RNA was obtained by in vitro transcription and then delivered into Huh7 cells by transfection. G418-resistant clones of Huh7 cells were obtained by selection. Clones of HCV full-length genome replication cell were confirmed by luciferase activity assay, Western blot and cleaveage of eYFP-MAVS by HCV NS3/4A protease. Then, HCV replication cell colonies were treated by different dose IFN-α in order to observe the change of luciferase activity, HCV protein and RNA level. Results At 3-4 weeks post-transfection, visible colonies were selected and stained by crystal violet. Luciferase activity and HCV NS3, NS5A protein were detected by luciferase activity assay and Western blot, respectively. Subcellular localization of eYFP-MAVS transferred from mitochondria to cytoplasms by cleavage of NS3/4A protease in cell colonies. Luciferase activity, HCV protein and RNA diminished obviously after IFN-α treatment. Conclusion A stable HCV full-length genome replication cell model labeled by reporter gene was successfully established and reporter activity can be used to indicate level of HCV proteins and RNA in cells. This cell model is a useful tool for the study on HCV pathogenesis and the screening of antiviral drugs.

Preparation of recombinant firefly luciferase by a simple and rapid expression and purification method and its application in bacterial detection / 西安交通大学学报·英文版

A simple and rapid expression and purification method of recombinant firefly luciferase was developed for bacteria detection. A modified luciferase gene from North American firefly Photinus pyralis was cloned into pET28a expression vector and the recombinant protein was produced in Escherichia coli BL21. The recombinant luciferase, equipped with a polyhistidine affinity tag, was purified by immobilized metal ion affinity chromatography (IMAC). The approach generated an abundant expression and an efficient purification of a recombinant luciferase with final yield 1.995 mg/L of cell culture. Experiments on the recombinant luciferase also showed that the relative light units (RUL) of the enzyme were 5.8×108, and the specific activity was 2.9×1010 RLU/mg. By applying adenosine triphosphate (ATP) bioluminescence to detection of the coin bacteria using the recombinant protein, the ATP content of bacteria was 9.48×10-16mol/mL, and was identical to the bacteria counts (4 500 CFU/mL) in order of magnitude. Taken together, our results provided a simple and efficacious method of the preparation of recombinant luciferase, which could be applied in the determination of bacteria via ATP bioluminescence.

Preparation of recombinant firefly luciferase by a simple and rapid expression and purification method and its application in bacterial detection / 药物分析学报

A simple and rapid expression and purification method of recombinant firefly luciferase was developed for bacteria detection. A modified luciferase gene from North American firefly Photinus pyralis was cloned into pET28a expression vector and the recombinant protein was produced in Escherichia coli BL21. The recombinant luciferase, equipped with a polyhistidine affinity tag, was purified by immobilized metal ion affinity chromatography (IMAC). The approach generated an abundant expression and an efficient purification of a recombinant luciferase with final yield 1.995mg/L of cell culture. Experiments on the recombinant luciferase also showed that the relative light units (RUL) of the enzyme were 5.8×108, and the specific activity was 2.9×1010 RLU/mg. By applying adenosine triphosphate (ATP) bioluminescence to detection of the coin bacteria using the recombinant protein, the ATP content of bacteria was 9.48×10-16mol/mL, and was identical to the bacteria counts (4500CFU/mL) in order of magnitude. Taken together, our results provided a simple and efficacious method of the preparation of recombinant luciferase, which could be applied in the determination of bacteria via ATP bioluminescence.

Bioluminescence Imaging of Chondrocytes in Rabbits by Intraarticular Injection of D-Luciferin / 핵의학분자영상

PURPOSE: Luciferase is one of the most commonly used reporter enzymes in the field of in vivo optical imaging. D-luciferin, the substrate for firefly luciferase has very high cost that allows this kind of experiment limited to small animals such as mice and rats. In this current study, we validated local injection of D-luciferin in the articular capsule for bioluminescence imaging in rabbits. MATERIALS AND METHODS: Chondrocytes were cultured and infected by replication-defective adenoviral vector encoding firefly luciferase (Fluc). Chondrocytes expressing Fluc were injected or implanted in the left knee joint. The rabbits underwent optical imaging studies after local injection of D-luciferin at 1, 5, 7, 9 days after cellular administration. We sought whether optimal imaging signals was could be by a cooled CCD camera after local injection of D-luciferin. RESULTS: Imaging signal was not observed from the left knee joint after intraperitoneal injection of D-luciferin (15 mg/kg), whereas it was observed after intraarticular injection. Photon intensity from the left knee joint of rabbits was compared between cell injected and implanted groups after intraarticular injection of D-luciferin. During the period of imaging studies, photon intensity of the cell implanted group was 5-10 times higher than that of the cell injected group. CONCLUSION: We successfully imaged chondrocytes expressing Fluc after intraarticular injection of D-luciferin. This technique may be further applied to develop new drugs for knee joint disease.

Monitoring of Electrophile Chemical Pollutants Using Transgenic Firefly Luciferase Gene Cells / 环境与健康杂志

Objective To assess quickly the potential environmental pollution by using the luciferase transgenic cells. Methods Many electrophile compounds in the environment can generate oxidative stress,so that the transcription of certain protective genes is induced via specific DNA motifs called electrophile response elements (EPREs). We have made a vector containing a single EPRE fused to the TK minimal promoter and the gene encoding firefly luciferase (EPRE-LUC) by adopting bio-molecular techniques. From this vector the stable LUC expression vector regulated by EPRE had been successfully reconstructed. This reporter construct was transfected into HeLa cells,and the clones resistant to G418 were selected. The resistant cells were treated by the different concentrations of sodium arsenate(NaAsO2),cadmium chloride (CdCl2),mercury chloride(HgCl2) and diethyl mateate (DEM). After that,the expression of luciferase was determined by luciferase assay kit. Results The correct construct frame of LUC reporter vector regulated by EPRE was identified by DNA sequencing; the dose-dependent relationships between the LUC expression and the test substance concentration were found. Among them,the relationship produced by DEM was the most significant. Conclusion The LUC transgenic cells regulated by EPRE have been successfully constructed.