Construction of recombinant plasmid pEGFP-NGB and its expression in cultured neuroglial cells

AIM: To establish recombination plasmid pEGFP-NGB and to investigate the expression of pEGFP-NGB in culture neuroglia cells. METHODS: The NGB cds was isolated by using RT-PCR method with total RNA extracted from fetal Kunming mouse brain, then the NGB cds was cloned into the eukaryotic expression vector pEGFP-C1 of EGFP reported green fluorescence protein. The expression vector of recombinant plasmid pEGFP-NGB was successfully constructed. GeneJamer transfection reagent was used to transfer recombinant plasmid pEGFP-NGB into culture neuroglial cells. The mRNA and protein expression of pEGFP-NGB in culture neuroglial cells were investigated. RESULTS: The positive clone sequencing was consistent with the sequence of Genbank. The NGB mRNA and protein expression of pEGFP-NGB in culture neuroglial cells were detected at high levels. The high expression of green fluorescence protein was observed by fluorescence microscope in culture neuroglial cells. CONCLUSION: The expression vector of recombinant plasmid pEGFP-NGB was successfully constructed and green fluorescence protein was expressed in cultured neuroglial cells.     

Construction of pNTAP-PRAK eukaryotic expression plasmid and its expression in HEK293 cells

AIM: To construct pNTAP-PRAK eukaryotic expression plasmid and to establish a stable HEK293 cell line expressing tandam affinity purification (TAP)-tagged PRAK. METHODS: Human PRAK coding region was subcloned into pNTAP vector to construct a recombinant plasmid called pNTAP-PRAK, then DH5α E.coli was transformed with the recombinant plasmid. After identified by PCR, digestion with restriction endonuclease and sequencing, the correct recombinant expression plasmid was transfected with PolyFect liposome transfection reagent to HEK293 cells. The cell line with stable expression of exogenous TAP tagged-PRAK gene was established by screening of antibiotic G418. The expression and localization of the fusion protein TAP tagged-PRAK were detected by Western blotting and immunofluorescence assay. RESULTS: All the results of identification by PCR, digestion with restriction endonuclease and sequencing indicated that the recombinant eukaryotic expression plasmid pNTAP-PRAK was constructed correctly. The result of Western blotting showed that the recombinant plasmid was expressed stably in HEK293 cells after transfection followed by G418 screening. The result of immunofluorescence assay showed that the expression product TAP tagged-PRAK distributed mainly in the nucleus. CONCLUSION: The eukaryotic expression vector pNTAP-PRAK was successfully constructed and the cell line stably expressing TAP tagged-PRAK was established. TAP tag didnt influence the localization of exogenous PRAK.     

Effect of adipose differentiation-related protein on proliferation and apoptosis in H9c2 cells

AIM: To construct an adipose differentiation-related protein (ADRP) eukaryotic expression vector and to explore the effect of ADRP on apoptosis of H9c2 cells induced by palmitic acid (PA). METHODS: The ADRP gene obtained by the method of RT-PCR was cloned into pEGFP-C1 plasmid. The recombinant plasmid was transformed into E.coli DH5α for amplification. The recombinant plasmid was extracted from E.coli DH5α and transfected into H9c2 cells by Lipofectamine^TM2000. The stable transformants were selected by G418 screening. Expression of green fluorescent protein was observed under fluorescence microscope and the ADRP expression was identified by RT-qPCR and Western blotting analysis. The effect of PA on the proliferation of H9c2 cells was detected by MTT assay. The apoptotic percentage of H9c2 cells caused by PA was determined by flow cytometry. RESULTS: The eukaryotic expression vector pEGFP-C1-ADRP was successfully constructed. Green fluorescent was observed in the cells transfected with pEGFP-C1 or pEGFP-C1-ADRP under fluorescence microscope. RT-qPCR and Western blotting analysis showed that recombinant cells exhibited high mRNA and protein levels of ADRP. After treated with PA at different concentrations, the apoptosis rates and the proliferation inhibition of recombinant cells were both lower than those of the other two cells. CONCLUSION: The transfected H9c2 cells with stable ADRP expression were successfully established. The over-expression of ADRP prevents the cells from apoptosis and inhibition of proliferation caused by PA, indicating that ADRP plays a protective role in H9c2 cells.     

Effect of human scavenger receptor SR-A I overexpression on uptaking ox-LDL and adhesion in 293T cells

AIM: To construct the recombinant eukaryotic expression plasmid pEGFP-C1-SR-A I for the high expression in 293T cells in order to identify functions of savenger receptor-A I (SR-A). METHODS: The primer was designed according to MSR1 cDNA and pEGFP-C1-SR-A I was constructed by standard molecular cloning technique and enzyme digestion. After sequencing, the plasmid was transfected into 293T cells by lipidosome method. The expression of scavenger receptor-A I was identified by RT-PCR and Western blotting. The foam cells were evaluated by the formation of lipid granules in the cells with oil red staining. Cell adhesion was analyzed by cell adhesion assay. RESULTS: 24 h after transfection, SR-A I mRNA was highly expressed and the high level of the protein was detected. The ratio of foam cell formation was doubled, the efficacy of cell adhesion was enhanced two times compared to the control group and the empty vector group. CONCLUSION: The recombinant eukaryotic expression plasmid has been constructed successfully with enhancing the function of uptake ox-LDL and adhesion in 293T cells by overexpression of SR-A I.     

Construction of mouse pdx-1 gene eukaryotic expression vector and its expression in embryonic stem cells

AIM: To clone mouse pdx-1 gene and construct its eukaryotic expression vector for expression of pdx-1 in mouse embryonic stem cells.METHODS: Mouse pdx-1 cDNA fragment was amplified with polymerase chain reaction (PCR) from mouse pancreatic cDNA. The purified fragment was recombinated with a eukaryotic expression vector carrying enhanced green fluorescent protein, pEGFP-N1. The pdx-1 cDNA fragment was inserted into the multi-clone sites of the vector to construct a new plasmid, pEGFP/pdx-1. E.colli strain DH5α was transfected with the new recombinant plasmid to expand it. Plasmid DNA extracted from the expanded DH5α was identifed by cutting with Hind Ⅲ, BamHⅠ nuclease and by DNA sequencing. Identified plasmid DNA was transfected into mouse embryonic stem cell line MESPU13 by carrying with liposome. RESULTS: A 876 bp cDNA fragment was amplified from mouse pancreatic cDNA by PCR and it was inserted into the vector pEGFP-N1 correctly. The fragment was defined to be pdx-1 gene by nuclease digestion and DNA sequencing. Mouse embryonic stem cell line MESPU13 was transfected with the new recombinant plasmid DNA. The green fluorescent protein report gene and pdx-1 gene expressed in transfected mouse embryonic stem cells within 24 h. CONCLUSION: Mouse pdx-1 gene is cloned and its recombinant eukaryotic expression vector carrying green fluorescent protein is constructed successfully. It provides a useful tool for further research on the function of pdx-1.     

Cloning and expression of mouse canstatin cDNA in E.coli

AIM: To clone and express mouse canstatin (m canstatin) cDNA and provide a basis for the further research on its anti-angiogenic activity and potential application for cancer therapy. METHODS: Total RNA was extracted from mouse liver tissue by Trizol Reagent, and mouse canstatin cDNA was amplified by RT- PCR, then cloned into vector pMD18-T for sequencing. pET30a(+)-m canstatin recombinant plasmid was constructed and expressed in E.coli BL21 with induction of IPTG. RESULTS: Mouse canstatin cDNA is 684 bp coding 227 amino acids. The sequences of both cDNA and amino acid share high homology with human canstatin, with cDNA identity at 89% and amino acids identity at 96% to human canstatin. In the present study, pET30a(+)-m canstatin recombinant plasmid was expressed in E.coli BL21. CONCLUSION: Mouse canstatin cDNA has been cloned for the first time. Constructed pET30a(+)-m canstatin recombinant plasmid is highly expressed in E.coli BL21.     

Construction of fusion expression vector AR-GFP and its expression in Hek293 cells

AIM: To construct a hAR and GFP fusion gene vector and to observe the AR-GFP gene expression in Hek293 cells. METHODS: A recombined vector pcDNA3.1/myc-HisA-AR-GFP (pH-AG) was constructed by gene engineering technique. The recombined vector was transfected into Hek293 cells using calcium phosphate. RESULTS: AR-GFP fusion protein was successfully expressed in Hek293 cells without biologic activity, which was confirmed by fluorescence microscopy and Western blotting. CONCLUSION: The Hek293 cells transfected by AR-GFP fusion gene can express its protein successfully. However, it is not a cellular model for ARI screening.      

Construction of eukaryotic expression vector of fusion gene CD80-IgG and its expression in Chinese hamster ovary cells

AIM: To construct the eukaryotic expression vector CD80-IgG by fusing the cDNA encoding extracellular portion of murine CD80 to the 5'-terminus of cDNA encoding Fc fragment of murine immunoglobulin G1 and to express the fusion protein in Chinese hamster ovary (CHO) cells. METHODS: The two cDNAs was amplified by PCR respectively from plasmid pcDNA/B7 containing the full-length cDNA of murine CD80 from murine spleen cells, and cloned to the eukaryotic expression vector pcDNA3.0 by directional cloning. The resultant recombinant plasmid pcDNA/CD80-IgG was transfected into CHO cells with liposome transfection reagent. The stably expressing cells were obtained by G418 screening. Western blot, Dot ELISA, and flow cytometry were used to detect the expression of the fusion protein and its immunological activity. RESULTS: DNA sequencing verified the correction of the construction of recombinant plasmid pcDNA/CD80-IgG. The expressed fusion protein was detected in the supernatant of transfected CHO cells and the molecular weight of the protein was similar to what we expected. Its immunological activity was also established. CONCLUSION: The recombinant plasmid pcDNA/CD80-IgG was successfully constructed and it expressed the fusion protein CD80-IgG.     

Construction of recombinant plasmid pEGFP-HSP70 and its expression in rat neural stem cells

AIM：To explore the method of constructing recombinant plasmid pEGFP-HSP70 and its expression in neural stem cells. METHODS：Total RNA was acquired from the fetal liver tissue of SD rat. cDNA complete sequence of heat-shock protein 70 (HSP70) gene was amplified by RT-PCR, and cloned into an eukaryotic expression vector containing the enhanced green fluorescent protein (EGFP) reporter gene, pEGFP-C2. Sequencing analysis was performed to confirm the recombinant plasmid pEGFP-HSP70. The technique of nucleofector transfection was used to transfect the recombinant plasmid pEGFP-HSP70 into neural stem cells. RESULTS：HSP70 cDNA sequence was correctly cloned into the eukaryotic expression vector pEGFP-C2. The recombinant plasmid pEGFP-HSP70 was constructed successfully. Compared with control group, the fluorescence intensities in pEGFP-C2 group and pEGFP-HSP70 group were significantly increased. The fluorescence intensity in pEGFP-HSP70 group after 24 h of transfection was significantly decreased compared with other time points of 7 d, 14 d and 21 d. The expression level of HSP70 significantly increased 1 d, 7 d and 14 d after transfection compared with control group. CONCLUSION：The neural stem cells can be directly used as gene action target cells. The HSP70 expression level in the stem cells is closely related to the time after transfection.     

Construction of human anti-HBc ScFv eukaryotic expression vector and expression of anti-HBc ScFv in HepG2 cells

AIM: To construct an eukaryotic expression vector of human single-chain variable fragment against hepatitis B virus core protein (anti-HBc ScFv) and detect its expression in HepG2 cells. METHODS: Anti-HBc ScFv genes were amplified from the plasmids abstracted from positive clone and inserted into pEGFP-c1 vector that contained green fluorescent protein gene. The recombinant plasmids were transfected into HepG2 cells, and resistant clones were obtained by G418 selection. The expression of the gene of fusion protein was determined by fluorescent invert microscope and ELISA. RESULTS: Recombinant plasmids were successfully constructed. The plasmid transfected HepG2 cells were obtained by G418 selection. Specific fluorescence was observed in HepG2 cells 48 hours after transfection. ELISA analysis confirmed the expression of anti-HBc ScFv in the cells. CONCLUSION: The construction of human anti-HBc ScFv eukaryotic expression vector and its expression in HepG2 cells lay the foundation for advanced research of intracellular anti-HBc ScFv.     

Effects of intracellular expression of Mycobacterium tuberculosis CFP10-ESAT6 fusion protein on proliferation and apoptosis of mouse macrophages

AIM：To establish Mycobacterium tuberculosis culture filtrate protein 10 (CFP10)-early secretory antigenic target 6 (ESAT6) eukaryotic expression vector and investigate the effect of intracellular expression of CFP10-ESAT6 fusion protein on the proliferation and apoptosis of mouse macrophages (RAW264.7 cells). METHODS：The recombinant plasmid pEGFP-N1/CFP10-ESAT6 was constructed by inserting CFP10-ESAT6 fusion gene into eukaryotic expression vector pEGFP-N1, and then transfected into RAW264.7 cells to express CFP10-ESAT6 fusion protein. The viability of RAW264.7 cells was measured by MTT assay. Flow cytometry was applied to measure the apoptotic rate and Toll-like receptor 2 (TLR2) expression in RAW264.7 cells treated with Mycobacterium tuberculosis 19 kD lipoprotein or staurosporine. RESULTS：The recombinant plasmid pEGFP-N1/CFP10-ESAT6 was successfully constructed and transfected into RAW264.7 cells. Compared with the control cells, intracellular expression of CFP10-ESAT6 fusion protein did not affect the viability of RAW264.7 cells, but could inhibit the apoptosis of RAW264.7 cells treated with Mycobacterium tuberculosis 19 kD lipoprotein. Moreover, CFP10-ESAT6-expressing macrophages had markedly lower expression of TLR2 on the surface. CONCLUSION：Intracellular expression of CFP10-ESAT6 fusion protein has no cytotoxicity on mouse macrophages, but can inhibit the apoptosis of the macrophages treated with Mycobacterium tuberculosis 19 kD lipoprotein through down-regulating the expression of TLR2.     

Construction of eukaryotic expression vector with EGFP and hVEGF121 fusion protein and its expression in mesenchymal stem cells of rats

AIM: To construct a recombinant plasmid carrying enhanced green fluorescent protein and human vascular endothelial growth factor 121 gene and to detect its expression in rats mesenchymal stem cells (MSCs). METHODS: Human VEGF121 cDNA was amplified with polymerase chain reaction (PCR) from pCD/hVEGF121 and was inserted into the eukaryotic expression vector pEGFP-C1. The recombinant plasmid pEGFP/hVEGF121 was identified with PCR, double enzyme digestion and DNA sequencing. Then this recombinant plasmid was transfected into rat's MSCs with lipofectamine. The expression of EGFP and VEGF121 protein were detected with fluorescence microscope and immunocytochemical staining, respectively. RESULTS: The recombinant plasmid was confirmed with PCR, double enzyme digestion and DNA sequencing. The fluorescence microscope and immunocytochemical staining results showed that the EGFP and VEGF121 protein were expressed in MSCs 48h after transfection. CONCLUSIONS: The recombinant plasmid carrying enhanced green fluorescent protein and human vascular endothelial growth factor was successfully constructed and expressed positively in rat MSCs. It provides a good basis for further research on differentiation of MSC and VEGF gene therapy for ischemial cardiovascular disease.     

Prokaryotic expression and functional study of human high mobil ity group box 1 protein

AIM:To construct the prokaryotic expressio n plasmid of His-tagged human high mobility group box 1 fusion protein (hHMGB1) and to express the fusion protein in E.coli for the affinity purification.METHODS:The cDNA coding region of HMGB1 was amplified by PCR fr om pGEX4T-HMGB1 and cloned into a modified pET14b vector following the routine p rocedure.After identification by enzyme digestion,PCR and sequencing,the plas mid was transformed into BL21 (DE3) competent cells,and the His-HMGB1 fusion pro tein was induced for expression with isopropyl-beta-D-thiogalactopyranoside (IPT G),and further purified by Ni-NTA affinity chromatography.The protein was f il tered for sterilization and used to stimulate human umbilical vein endothelial c ells (HUVECs).24 hours later,the cultured supernatant of HUVECs was collected for the detection of cytokines/chemokines with LiquiChip system.RESULTS:The His-HMGB1 fusion protein expression plasmid was ide ntified by enzyme digestion and sequencing.The purified His-tagged fusion prote in was analyzed by SDS-PAGE and Western blotting with specific anti-His antibody .It was found that the production of IL-8 from HUVECs was highly induced in a d ose-dependent manner by HMGB1.CONCLUSION:The His-tagged HMGB1 fusion protein expression plasm id was successfully constructed,and purified.Recombinant HMGB1 protein has a h igh bioactivity on the induction of cytokines in HUVECs,which may significantly facilitate the future study of HMGB1 biological functions.     

欢迎订阅2006年下列期刊

AIM: To construct prokaryotic expression vector of His-tagged human IP-10 for further study of its biological function in the inflammatory response. METHODS: The coding sequence of IP-10 lacking signal peptide was amplified from human lung cDNA library by polymerase chain reaction (PCR) and the fragment was cloned into pET-14b plasmid for the construction of His-tagged fusion protein expressing vector, pET-14b/IP-10. After being identified by enzyme digestion and sequencing, the recombinant vector was transformed into a strain of E. coli, BL21 (DE3). The expression of His-tagged fusion protein was induced with IPTG and purified with Ni+-NTA affinity chromatography. Then the chemotactic activity of IP-10 was determined by transwell migration assay on THP-1 cells. RESULTS: The construction of pET-14b/IP-10 recombinant vector was proved by enzyme digestion and sequencing. The fusion protein IP-10, which was purified by a routine Ni+ affinity method, had an activity on the induction of cell migration of THP-1. CONCLUSION: We successfully construct IP-10 fusion protein expressing vector and get the fusion protein with high bioactivity, which provides essential materials for the future studies on IP-10. V     

Subcellular localization of ZNF580-EGFP fusion protein

AIM: To construct a eukaryotic expression plasmid for enhanced green fluorescent protein (EGFP) and ZNF580 fusion protein, and study its subcellular localization in the transfected MGC803 cells. METHODS: The primers were designed according to the cDNA encoding sequence of ZNF580 full-length open reading frame (1-172aa), ZNF580 amino terminus (1-93aa) and ZNF580 carboxyl terminus (94-172aa). The three cDNA segments of PCR were cloned into pGEM-T vector. Then they were subcloned respectively into plasmid pEGFP-C1 (enhanced green fluorescent protein). The subcellular localization of the fusion protein in MGC803 cells transfected with the vector was monitored by autofluorescence microscopy. RESULTS: Restricted enzymes analysis and DNA sequencing showed that the sequences of the pEGFP-ZNF580 (1-172), pEGFP-ZNF580 (1-93) and pEGFP-ZNF580 (94-172) transgenic plasmid were correct. The fusion proteins of EGFP-ZNF580 (1-172) and pEGFP-ZNF580 (94-172) were localized in the nuclei. CONCLUSION: The recombinant eukaryotic expression vector pEGFP-ZNF580 has been successfully constructed. The nuclear localization signal is within amino acid residues 94 and 172 of ZNF580 carboxyl terminus (C2H2 zinc finger domain).     

Construction and expression of recombinant adenovirus vector encoding human somatostatin receptor type 2

AIM: To construct recombinant adenovirus vector carrying the gene of human somatostatin receptor type 2 （SSTR2） for gene therapy of pancreatic carcinoma.METHODS: SSTR2 cDNA was inserted into adenovirus shuttle plasmid pDC316, named pDC316-SSTR2.pDC316-SSTR2 was cotransfected with rescue plasmid pBHGlox (delta) E1, 3Cre into 293 cells by liposome reagent.Ad-SSTR2 was generated by site-specific recombination and confirmed by PCR.Ad-SSTR2 was propagated in 293 cells and purified.The titer of viral stock was determined by end-point dilution assay.Western blotting was used to determine the expression of SSTR2 protein after human pancreatic carcinoma cell capan-2 was infected with recombinant adenovirus.RESULTS: pDC316-SSTR2 was successfully constructed.Recombinant adenovirus Ad-SSTR2 was acquired by pDC316-SSTR2 and pBHGlox (delta) E1, 3Cre cotransfected into 293 cells.Ad-SSTR2 was characterized by PCR.The virus titer was 6.0×1012 pfu/L.SSTR2 protein was detected after adenovirus infected capan-2 48 h with Western blotting.CONCLUSION: The recombinant adenovirus vector encoding human SSTR2 is successfully constructed and correctly expressed in pancreatic carcinoma cells.This investigation provides the basis for study of gene therapy of pancreatic carcinoma.     

Construction of the vector for fusion protein gene driven by IGF-Ⅱ P3 promoter and its expression in hepatocellular carcinoma cells

AIM:To construct the shuttle plasmid vector for thymidine kinase(tk) and EGFP fusion protein gene driven by IGF-Ⅱ P3 promoter,and investigate the specific killing effect of the HSV-tk/GCV system on hepatocellular carcinoma(HCC) cells in vitro.METHODS:Recombinant shuttle plasmid vector was constructed by techniques of genetic recombination and screening,and identified by restriction digestion and sequencing analysis. Then the recombinant shuttle plasmid was transfected into HepG2 and HeLa cells by techniques of lipofectamine transfection and its expression was detected by fluorescence microscope and RT-PCR. Cell killing after ganciclovir(GCV) application was determined by MTT.RESULTS:Identification of pDC316-tkEGFP-P3 by enzyme digestion and sequencing analysis showed that the length,inserted location and direction of the target genes which were inserted into the recombinant were correct. It was found that enhanced green fluorescence protein could only be seen in HepG2 cells,but not in HeLa cells. The results of RT-PCR showed that only two bands could be seen in the samples of pDC316-tkEGFP-P3 transfected HepG2 cells. The MTT test showed the selective cytotoxicity of GCV to the transfected HepG2 cells.CONCLUSION:The shuttle plasmid vector carrying the tkEGFP fusion protein gene driven by IGF-Ⅱ P3 promoter has been constructed successfully and its specific expression in HepG2 cells provided a sound basis for targeted gene therapy for HCC.