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Objective: To produce fluorescent tagged recombinant erythroferrone protein (ERFE-eGFP) for laboratory investigations. Methods: Erythroferrone (ERFE) gene was fused to green fluorescent protein (eGFP) gene and cloned in a pSecTag2Hygro plasmid. The constructed plasmid was amplified in Escherichia coli DH5 α and the eGFP-fused ERFE (ERFE-eGFP) protein was expressed in human embryonic kidney (HEK293T) cell line. Results: The plasmid constructed from colony C6 contained ERFE-eGFP with the correct restriction sizes of 4.2 kb and expressed secretory ERFE-eGFP fusion protein (approximately size of 75 kDa) in HEK293T cell line. Conclusions: ERFE-eGFP recombinant protein is successfully expressed as a secretory functional protein and could be sensitively detected using fluorometry. This fusion protein might benefit future applications for localization of cellular ERFE receptors and competitive immunoassay of ERFE concentration.
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@# Objective: To investigate the relationship between long non-coding RNA (lncRNA) HIT and cisplatin (DDP) resistance in osteosarcoma cells and the mechanism related to epithelial-mesenchymal transition (EMT). Methods: 42 pairs of osteosarcoma tissues and corresponding para-cancerous tissues (more than 5 cm away from the edge of cancer tissues) were collected at the Department of Orthopedics, Tianjin Hospital during June 2017 to June 2018. Quantitative Real-time PCR (qRT-PCR) was used to detect the mRNAexpression of HIT and EMT related markers (Snail and E-cadherin) in the collected tissues. The DDP-resistant osteosarcoma U2OS cell line was constructed and human adrenal 293T cell line was used as control. Two sets of siRNA vectors targeting HIT loaded on lentivirus were transfected into cells with DDP-resistance as the interference group A and group B. Meanwhile, the U2OS cell line was transfected with HIT full-length vector and blank vector respectively, as over-expression group and blank group. The DDP 50% inhibitory concentration (IC50) was detected by MTT assay. qRT-PCR was used to detect the mRNA expressions of HIT, Snail and E-cadherin. Western blotting was used to detect the protein expressions of Snail and E-cadherin. RNA binding protein immunoprecipitation (RNAIP) assay was used to clarify the combination of HIT and Snail protein in the U2OS and 293T cells. Results: The mRNAexpressions of HIT and Snail in osteosarcoma tissues were significantly higher than those in para-cancerous tissues, while the mRNA expression of Ecadherin was significantly lower than that in the paracancerous tissues. The mRNA expression of HIT and E-cadherin in osteosarcoma tissues was negatively correlated (all P<0.01). The DDP IC50 in the DDP-resistance group was significantly higher than that in the control group, interference group A and B, and the DDP IC50 in over-expression group was significantly higher than that in blank group (all P<0.01). The expression of HIT in resistance group was significantly higher than that in the control group, and the HIT expressions in interference group A and B were significantly lower than that in DDP-resistance and control group; moreover, the expression of HIT in over-expression group was significantly higher than that in blank group (all P<0.05 or P<0.01). The mRNAexpression of Snail in DDPresistance group was significantly higher than that in the control group and interference group A and B, while the mRNA expression of E-cadherin in DDP-resistance group was significantly lower than that in the control group and interference group A and B; and the mRNA expression of E-cadherin in over-expression group was significantly lower than that in blank group. The protein expression of Snail in the DDP-resistance group was significantly higher than that in the control group and interference group A and B,while E-cadherin protein expression was significantly lower; and protein expression of Snail in over-expression group was significantly higher than that in blank group (all P<0.05 or P<0.01). The expression of HIT in the U20S and 293T cells treated by anti-Snail antibody induced by immunomagnetic beads was significantly higher than that in the cells treated by IgG antibody (P<0.01). Conclusion: HIT can promote EMT and cisplatin-resistance in osteosarcoma cells through up-regulation of Snail protein and inhibition of E-cadherin transcription activity.
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Objective: To produce fluorescent tagged recombinant erythroferrone protein (ERFE_eGFP) for laboratory investigations. Methods: Erythroferrone (ERFE) gene was fused to green fluorescent protein (eGFP) gene and cloned in a pSecTag2Hygro plasmid. The constructed plasmid was amplified in Escherichia coli DH5α and the eGFP-fused ERFE (ERFE_eGFP) protein was expressed in human embryonic kidney (HEK293T) cell line. Results: The plasmid constructed from colony C6 contained ERFE_eGFP with the correct restriction sizes of 4.2 kb and expressed secretory ERFE_eGFP fusion protein (approximately size of 75 kDa) in HEK293T cell line. Conclusions: ERFE_eGFP recombinant protein is successfully expressed as a secretory functional protein and could be sensitively detected using fluorometry. This fusion protein might benefit future applications for localization of cellular ERFE receptors and competitive immunoassay of ERFE concentration.
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Objective To knockout the exon51 of DMD gene in HEK293T cells using the CRISPR/Cas9 system. Methods Design the target sequences of sgRNA and clone them into plasmid PX459 respectively; transfer these plasmids into HEK293T cell and extract the total genome DNA; test the activity of sgRNAs with surveyor assay, choose the most efficient one in each end;construct plasmid PX459-2sgRNA and transfer it into HEK293T cells;check whether the exon51 has been knocked known with PCR and T vector sequencing. Results 50% of HEK293T cells' DMD gene exon51 were knocked out,showing a high gene editing efficiency. Conclusions We successfully establish a platform to target knockout the exon51 of DMD gene and provide an important experimental basis for the treatment of DMD and other genetic diseases.
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Objective:To construct lentiviral vector which can overexpression miR-137 and produce lentivirus by lentivirus packaging system, and to explore its infection efficiency and expression in HEK293T cells.Methods: miR-137 sequence was chemically synthesized and cloned into lentiviral vector GV209, and the recombinant plasmid containing human miR-137 was obtained and identified.Then miR-137 recombinant plasmid together with two helper plasmids were transfected into HEK293T cells using Lipofectamine 2000.After the HEK293T cells were infected in multiplicity of infection(MOI) 40 for 48 h, the expression of green fluorescent protein (GFP) was observed by fluorescence microscope and the expression level of miR-137 was detected by fluorescence quantitative PCR.Results:The sequencing results showed that the inserted gene sequence was completely consistent with the published human miR-137 gene sequence in GenBank.The GFP was observed in the HEK293T cells infected with miR-137 overexpression lentivirus under fluorescence microscope.The fluorescence quantitative PCR results showed that the expression level of miR-137 in the cells infected with overexpression lentivirus was 12.74 times higher than that in the control cells.Conclusion:The lentivirus containing miR-137 gene is successful packaged, and it could efficiently infect the HEK293T cells.
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Objective To prepare recombinant human prothrombin-2 expressed in Pichia pastoris, and assay the enzymatic and clotting activities of prothrombin-2 activated by prothrombin activator ecarin.Methods Human prothrombin-2 gene and Echis carinatus ecarin gene were synthesized separately on the basis of the cDNA sequences published in GenBank.The gene of prothrombin-2 was cloned into the expression vector pPICZαA.The expression vector pPICZαA/prothrombin-2 was transformed into glycoengineered P.pastoris, and then prothrombin-2 engineered P.pastoris was screened.The expression products were induced by methanol, purified by two-step chromatography and identified by diges-tion by PNGase F and analysis of pepetide fingerprint.The ecarin gene was cloned into the expression vector pcDNA3.1. The expression vector pcDNA3.1/Ecarin was transformed into HEK 293T cells and the culture supernatant of HEK 293T/Ecarin was collected.The reaction product of HEK 293T/Ecarin cell culture supernatant and purified prothrombin-2 was analyzed by S-2238,which was the chromogenic substrate for thrombin.Fibrinogen was used to measure blood clotting time. Results The purified protein of P.pastoris expressed prothrombin-2 culture supernatant was 37 ×103 .The relative molecular mass(Mr) of the purified protein was reduced to 35 ×103, which was consistent with the theoretical Mr of prothrombin-2 molecular weight.The purified protein was proved to be prothrombin-2 by peptide fingerprint identification. The purified prothrombin-2 processed by HEK 293T/Ecarin culture supernatant could hydrolyze S-2238 to produce yellow pNA, and D405 of pNA increased with the volume of the processed prothrombin-2 that could promote the plasma coagulation.The blood clotting time was close to that of the thrombin kit.Conclusion Prothrombin-2 is prepared by P.pastoris and activated toα-thrombin by ecarin.This technique may replace the method of extraction of prothrombin from plasma and can be used for the treatment of war wounds or for future clinical research.
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Objective To establish a cell line stably expressing the human CD36 by using TA clo-ning and cell transfection technology and to analyze its application to the detection of anti-CD36 antibodies. Methods Total RNA was isolated from human platelets and then used to synthesize complementary DNA ( cDNA) . Sequence of the gene encoding CD36 on human platelets was obtained by PCR amplification. The recombinant vector was transformed into TOP10 E. coli after TA cloning. The positive recombinant pcDNA3. 1/V5-CD36 plasmid was screened out by blue-white selection and then sequenced. The correctly constructed plasmid coated with Effectene? Transfection Reagent was transferred into HEK293T cells. Fluo-rescence-activated cell sorting was performed to screen out the cell line that could stably express the CD36 on human platelets. The transfected cell line-based flow cytometry analysis and antibody capture assay ( ACA) were established and used for antibody detection in nine serum samples positive for anti-CD36 antibodies. Results The HEK293T cell line stably expressing the recombinant CD36 was successfully established. Compare with the monoclonal antibody immobilization of platelet antigens assay ( MAIPA) , anti-CD36 anti-bodies could be easily identified in nine serum samples by using the transfected cell line-based flow cytome-try analysis and ACA. Conclusion This study suggests that the HEK293T cells stably expressing the re-combinant CD36 could be used in flow cytometry analysis and ACA for the detection of anti-CD36 antibod-ies. It also paves the way for further researches on the mechanism of CD36 in other diseases.
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Objective To construct and identify lentiviral vector pGC-FU-CXCR4 gene. Methods CXCR4 gene amplification was used by real-time polymerase chain reaction. The target gene fragments with the digested plasmids were exchange. Then the lentiviral vector pGC-FU-CXCR4 was constructed successfully. Use the constructed lentiviral vector to infect the competent escherichia coli cells. Polymerase chain reaction analysis was used to identify the cultural clones and DNA sequencing and comparative analysis were used to positive fragments. The successfully constructed plasmids had the same sequence with the target gene. Results Polymerase chain reaction tests showed that am-plified target genes were inserted in pGC-FU vectors. The electrophoresis results,digestion showed that the reconstructed plasmid was consist-ent with the theoretical fragment and the sequence result of the positive fragments were exactly the same with the target gene. Conclusion Lentiviral vectors of CXCR4 gene over-expression were successfully constructed.
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Objective To construct a recombinant lentiviral vector for human thymosin ?4 (TMSB4) and to test the mRNA and protein expression of TMSB4 in 293T cells after being infected by shRNA lentivirus. Methods We designed a specific sequence of small hair RNA targeting TMSB4 gene; the complementary DNA containing both sense and antisense oligo DNA of the targeting sequence was cloned into the pGCSIL-GFP vector to construct a lentiviral vector. The vector was converted into the competent DH5a coli,which confirmed by PCR and sequencing. Then the viral vector and the packed systemic vector were cotransfected 293T cells to get the lentivirus. The virus titer was determined. Afterwards,in the 293T cells infected with the lentivirus,the expression of TMSB4 was detected by real time PCR and immunocytochemistry. Similarly,a primary fibroblast was also infected with the lentivirus. Results Compared with negative control cells,the mRNA and protein levels of TMSB4 in 293T cells infected with the lentivirus were reduced by 44% (0.56?0.11 vs 1.00?0.06,F=89.673,P
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Objective To clone the full length cDNA of human LIGHT and to construct the recombinant eukaryotic expression plasmid pCI neo LIGHT for the stable expression on 293T cells. Methods Human LIGHT cDNA was cloned from a normal human activated T cell cDNA library phAD.CAD by PCR. After sequencing, LIGHT cDNA was inserted into plasmid pCI neo for the construction of the eukaryotic expression. The recombinant was transfected into 293T cells by electroporation. The expression of LIGHT on the surface of 293T cells was detected by flow cytometry after screening with G418. Results Sequencing confirmed that ORF of LIGHT gene was intact and right. Restrictive enzyme digestion proved that LIGHT gene was inserted into the recombinant plasmid of LIGHT pCI neo correctly. FACS analysis revealed that about 78.69% 293T cells expressed LIGHT protein on the cell surfaces at 3 months after screening with G418. Conclusion LIGHT gene has been cloned successfully, and a 293T cell line expressing LIGHT protein on its membrane surface has been obtained.