Construction of foot-and-mouth disease virus like particles-induced expression vectors and screening of BHK-21 cell pools / 生物工程学报

Transient expression is the major method to express foot-and-mouth disease virus (FMDV) capsid proteins in mammalian cells. To achieve stable expression of FMDV capsid proteins and efficient assembly of virus like particles (VLPs) in cells, the plasmids of piggyBac (PB) transposon-constitutive expression and PB transposon-tetracycline (Tet) inducible expression vectors were constructed. The function of the plasmids was tested by fluorescent proteins. By adding antibiotics, the constitutive cell pools (C-WT, C-L127P) expressing P12A3C (WT/L127P) genes and the inducible cell pools (I-WT, I-L127P) expressing P12A3C (WT/L127P) genes were generated. The genes of green fluorescent protein, 3C protease and reverse tetracycline transactivator (rtTA) were integrated into chromosome, which was confirmed by fluorescence observation and PCR testing. The cell pool I-L127P has a stronger production capacity of capsid proteins and VLPs, which was confirmed by Western blotting and enzyme linked immunosorbent assay (ELISA), respectively. In conclusion, inducing the chromosomal expression of FMDV capsid proteins was firstly reported, which may facilitate the technical process of mammalian production of FMDV VLPs vaccine and the construction of mammalian inducible expression systems for other proteins.

Construction and expression of the recombinant plasmids of truncated and normal selenoprotein S genes / 西安交通大学学报(医学版)

Objective To construct the recombinant plasmids of normal and truncated selenoprotein S genes so as to observe their biological function in vitro or in vivo.Methods We constructed the recombinant plasmids of normal and truncated selenoprotein genes by gene recombinant technology.The gene of truncated selenoprotein was coding domain sequence(CDS)fragment of mRNA;the gene of normal selenoprotein was CDS and 3'untranslated region(including Sec insertion sequence)fragment of mRNA.We confirmed the sequence of recombinant genes by sending them to a company for comparison.The recombinant plasmids of normal and truncated genes of SelS were transfected into cells by Lipofectamine 2000.After 24 hours,the expression of green fluorescent protein was observed and transfection efficiency was detected by FACS analysis.We collected the cells to isolate the total RNA by TRIzol method,and then cDNA was obtained by mRNA reverse transcription and amplified by PCR.Results The sequencing results showed that the recombinant genes were completely the same as the target genes,indicating that we constructed the plasmids successfully.The expression of green fluorescent protein could be observed and transfection efficiency was detected up to 40% by FACS analysis.PCR results showed that the target selenoprotein gene was highly expressed in the experimental group than in control group.Conclusion The truncated and normal selenoprotein S genes were successfully constructed and transfected into cells where they were highly expressed.It lays foundation for observing the biological effect of truncated and normal selenoprotein in cell line or animal body.

Construction and expression of multi-gene recombinant plasmid pEG-FP-N1-HBsAg-ROP2 / 中国血吸虫病防治杂志

Objective To construct pEGFP-N1-HBsAg-ROP2 recombinant expression plasmid and transfect HEK293T cells for expression,and pay a way for Toxoplasma gondii nucleic acid vaccine development. Methods According to the HBsAg gene sequence and pcDNA3-p30-ROP2 recombinant plasmid restriction sites,the HBsAg gene was amplified by PCR.The HB-sAg gene was cloned into the pcDNA3-p30-ROP2 and instead of p30 gene.The HBsAg-ROP2 fragment was amplified by PCR and digested with HindⅢand KpnⅠto clone into the pEGFP-N1 eukaryotic expression vector and construct the recombinant pEGFP-N1-HBsAg-ROP2.The expression vector was transfected into HEK293T cells based on the identification of PCR amplifi-cation,restriction endonucleases and sequencing.Results The PCR product of HBsAg was about 700 bp,which was consis-tent with the theoretical value.Two bands of about 5.4 kb and 1.9 kb were obtained after double enzyme digestion with pcDNA3-HBsAg-ROP2 recombinant plasmid.The recombinant plasmid pEGFP-N1-HBsAg-ROP2 was double-digested to generate an empty vector fragment of about 4.7 kb and a band of about 1.9 kb of HBsAg-ROP2 fragment.The results of sequencing showed that the sequence was 99.84% identical with the published sequence in GenBank.The target plasmid was successfully transfect-ed into HEK293T cells,and the expression was correct,the protein concentration was 3.08 mg/ml.Conclusion The recombi-nant plasmid pEGFP-N1-HBsAg-ROP2 is successfully constructed and expressed efficiently.

Construction and identification of eukaryotic expression plasmid carrying hTERT-P2A-EGFP / 吉林大学学报(医学版)

Objective:To construct the eukaryotic expression plasmid carrying hTERT-P2A-EGFP, and to explore its expression and transfection efficiency in the HEK293FT cells.Methods:The recombinant plasmid was constructed by using pBABE-puro-hTERT and pRRLSIN-cPPT-MSCV-EGFP plasmids.The hTERT,P2A,and EGFP genes were obtained using pBABE-puro-hTERT as template by PCR.And the correct hTERT was inserted into pRRLSIN-cPPT-MSCV-EGFP vector.Then the recombinant plasmid containing hTERT-P2A-EGFP gene was obtained and identified.The HEK293FT cells were transfected by the recombinant plasmid, and the expression of green fluorescence protein(GFP) was observed by fluorescence microscope.Results:The PCR results showed that the fragments of hTERT, P2A, and EGFP were 3 400, 110 and 720 bp.And the length of gene fragment(hTERT-P2A-EGFP)was 4 300 bp by enzyme digestion.The results of sequencing showed that the 1 547 site of the target gene was mutated.Using site-directed mutagenesis, the 1 547 site was successfully mutated.And the target gene sequence was completely identical with the sequence published in GenBank.The recombinant plasmid was transfected into the HEK293FT cells, and GFP was observed in the cells.The results of flow cytometry showed that the transfection efficiency of recombinant plasmid was 44.8%.Conclusion:The recombinant plasmid carrying hTERT-P2A-EGFP gene is successfully constructed, and it can be used for cell transfection.

Construction of the full length and N-terminal fragment of serum response factor over-expressing lentiviral plasmid and its impacts on cardiac stem cell differentiation / 临床与实验病理学杂志

Purpose To analyze the effects of full length and N-terminal fragment of serum response factor (SRF-Full and SRF-N) on TGF-β1-induced differentiation in c-Kit + cardiac stem cells (CSC).Methods Rat SRF-Full and SRF-N (1-254 aa) coding sequences were obtained from cDNA library and cloned into the linearized lentviral vector GV358 (Ubi-MCS3FLAG-SV40-EGFP-IRES-puromycin) to generate the recombinant vectors,and then positive clones were selected and sequenced after transducing the competent cells with recombinant vectors.The recombinant lentvirus were packaged through transfecting the HEK293T cells with SRF-Full,SRF-N overexpressing plasmids and viral packaging plasmids.Neonatal SD rat cKit + CSCs were isolated via magnetic activated cell sorting,and TGF-β1-induced differentiation in SRF-Full and SRF-N overexpression virus-infected CSCs was assessed by quantitative PCR.Results SRF-Full and SRF-N coding sequences were successfully obtained and properly cloned into the linearized GV358.The positive clones were selected and further confirmed by sequencing.With the help of packaging plasmids,the SRFFull and SRF-N overexpressing plasmids-transfected HEK293T cells successfully produced the lentiviral particles with the titer of 2 × 108 TU/mL,and the SRF-Full-Flag and SRF-N-Flag fusion protein were detected by Western blot in virus-infected HEK293T cells.Addition of TGF-β1 significantly induced upregulated mRNAs in cardiomyocyte markers (Nkx2.5,Gata4,cTnI) and smooth muscle cell marker (SM22α) but not the epithelia cell marker (vWF) in CSCs.Overexpression of SRF-Full facilitated TGF-β1-triggered cardiomyocyte differentiation.However,SRF-N exerted anti-differentiation effects in TGF-β1-treated cells.Conclusion The SRF-Full and SRF-N overexpressing recombinant lentiviral vectors are successfully constructed.SRF-Full facilitates while SRF-N suppresses TGF-β1-induced cardiomyocyte differentiation in c-Kit + CSCs.

Construction and functional identification of rat Hes1 adenovirus expression vector / 重庆医学

Objective to construct the high titers rat Hes1 adenovirus expression vector (Ad-Hes1).Methods With the rat cDNA as a template,the Hes1 fragment was amplified by PCR,which constructed pShuttle-CMV-Hes1 shuttle plasmid by directly clone.Based on pShuttle-CMV-Hes1,pAdeno-Hes1 virus plasmid was constructed,pAdeno-Hes1 was transfected into 293 cells to package Ad-Hes1,virus titers were determined by modified TCID50.Hes1 was detected by Western blot after Ad-Hes1 infected with H9c2 myocardial cells.Results pShuttle-CMV-Hes1 shuttle plasmid and pAdeno-Hes1 plasmid were constructed successfully,with a general titer of 1.6 × 1011 PFU,Ad-Hes1 can be expressed in H9c2 myocardial cells,and its MOI value was 30.Conclusion Ad-Hes1 is successfully constructedand packaged,thus provide basis for further research on the protection effect of Hes1 on myocardium.

Cloning short gene fragment and constructing recombinant plasmid based on restoration of antibiotic resistance / 生物工程学报

Molecular cloning is one of the most important and widely used technologies in molecular biology research. Generally, the target DNA fragment and the vector are separately digested by restriction enzyme, then purified and recovered, and then ligated with DNA-ligase. For some very short gene fragments (<300 bp), the recovery efficiency of the purified fragment is very low after digestion and cleavage, leading to the difficulty in its inserting into the expression vector. To address this issue, we developed a cloning method based on restoration of antibiotic resistance in constructing recombinant plasmid, which proved highly efficient in cloning very short gene fragments.

Establishment and optimization for TALE-TFs construction / 重庆医学

Objective To optimize the method of transcription activator‐like effector transcription factors (TALE‐TFs) con‐struction ,some improvement and adaption were made based on the traditional methods .Methods We first constructed the basic tandem fragments with different length ,including trimer ,tetramer ,pentamer and hexamer by Golden Gate cloning technique and PCR ,then the procedure with the highest efficacy was chosen to construct our TALE‐TFs .To determine the function of the TALE‐TFs ,the plasmid pminCMV with the specific binding sequence of TALE‐TFs was constructed by fragment substitution reaction (FSR) .The transcription activating function of TALE‐TFs was confirmed by the intensity of red fluorescence ,after TALE‐TFs , pEGFP‐N1 and pminCMV plasmid were co‐transfected into 293HEK cells .Results An optimized method for TALE‐TFs construc‐tion and functional assay was established .Conclusion This method can potentially be wildly used in fields that the expression of some constitutively expressed genes needs to be modified .

Mortalin promotes ovarian cancer cell growth through MAPK-ERK signal pathway / 解剖学报

Objective By constructing mortalin stably expressing ovarian cancer cell lines in A 2780 and A2780/cis, we demonstrate the role of mortalin in the ovarian cancer cell growth .Methods CCK-8 assay was used to measure cell viability in the overexpression mortalin group compared with the control group .The flow cytometry analysis was used to understand the effect of upregulated mortalin on the ovarian cancer cell cycle .Western blotting was used to determine the expression and phosphorylation level of MAPK /ERK and JNK/SAPK signal pathways .Results The results showed that increased expression of mortalin could accelerate ovarian cancer cell proliferation and promote G 1 transition, leading to a faster restoration of normal distribution of cell cycle .We found that mortalin overexpression significantly activated p-Raf and p-ERK1/2, but not p-JNK.Conclusion The results demonstrate that mortalin effect on the ovarian cancer cell proliferation contributes to active the MAPK-ERK signaling pathway .

Adenovirns construction with miceCD40- IgG2aFc-IRS-GFP fusion gene and in vitro testing / 中华肝胆外科杂志

Objective To construct the adenovirus vector containing mice CD40- IgG2aFc-IRS-GFP fusion gene and detect the expression of the fusion protein in transfeete 293cells.Methods The fragments of CD40, IgG2aFc were obtained and inserted into plasmid PDC316-IgG2aFc-GFP.After being verified by sequencing, Ad5-PDC316-CD40-IgG2aFc-GFP was contransfored with the bckbone vector into 293 cells.The virus titer was detected after replicating and purifing and the expression of the fusion protein was analyzed.Results A virus and plasmid were constructed successfully.The vitro infection showed that the virus can infect cells of which the fusion protein was confirmed by fluo-rescence.The expression of the fusion protein increased with the increased time and virus concentra-tion.The protein expression stopped increasing after the virus concentration came to a certain level.Conclusion CD40, IgG2aFc fragments are correctly ligated with plasmid PDC316-IgG2aFc-GFP, and the fusion protein can be expressed in 293cells.This might lay a foundation for further studies of the expresstion of virus, immune tolerance and mechanism of liver transplantation model in rats.

Plasmid construction and idetification of human vascular endothelial growth factor receptor(VEGF-R3) extracellular domain 1-3Loop cDNA abstract / 肿瘤研究与临床

Objective To insert VEGF-R3(FIT-4)gene fragment into cloning vector and identify it.Methods By T-A cloning,the VEGF-R3 gene fragment amplified by RT-PCR was cloned on the PGEM-T Easy plasmid vector and transfected into E.coli JM109.the recombinant clones were screened by"white-blue plaque selection" and tested by the methods of single restrictional enzyme.Results The VEGF-R3 gene fragment amplified by RT-PCR showed a smear after the electrophoresis on agarose Gel and obtained white clones by"white-blue plaque selection".The electrophoresis result of the recombinant clones tested by the single restrictional enzyme was accordant with expectant result.Conclusion The recombinant clones through the methods T-A may be used in the further gene expression and gene therapy studies.

Construction of pGL3-EPO-HRE reporter gene vector and detection of its function / 基础医学与临床

Objective To construct pGL3-EPO-HRE reporter gene vector and to detect its function.Methods To synthesize two single-strand DNA fragments which had overlap sequence at the 3'end,then mixed them to construct the double-strand DNA fragment which include three copies of(57 bp) human erythropoietin(EPO) HRE.There was MluⅠor BglⅡ digestive site at the end of this DNA fragment.After inserting the(87 bp) fragment into pMD18-T for sequencing,the correct sequence was inserted into pGL3-promoter to construct pGL3-EPO-HRE.The recombinant vector was co-transfected into MCF-7 cells with pcDNA3-HIF-1?or pcDNA3 respectively,(12 h) later cells were treated with(100 ?mol/L) cobalt chloride(CoCl_(2)) for(24 h), then the expression of pGL3-EPO-HRE was observed by dual-luciferase reporter gene assay method.Results The sequence of pGL3-EPO-HRE was identical with human EPO HRE sequence.pGL3-EPO-HRE luciferase activity increased about 10 fold(P

Construction of pcDNA3-HIF-1? eukaryotic expression vector and its expression in MCF-7 cells / 基础医学与临床

Objective To construct pcDNA3-HIF-1? eukaryotic expression vector and to investigate its function in breast cancer MCF-7 cells.Methods RT-PCR was applied to amplify human HIF-1? cDNA from MCF-7 cells with a pair of sequence specific primers carrying a restriction enzyme site XbaⅠ or HindⅢ on each 5′end.HIF-1?(cDNA) was inserted into pMD18-T after sequencing,and then inserted into pcDNA3 vector.The recombinant vector was transfected into MCF-7 cells to observe its expression and function by Western blot and dual-luciferase reporter gene assay.Realtime-PCR was performed to detect the inducible expression of C-MET mRNA by HIF-1?,the anti-apoptotic effect of HIF-1 under hypoxia was analysed by detecting the Caspase3/7 activity.Results The sequence of pcDNA3-HIF-1? is identical with the gene bank.It enhances the expression of HRE reporter gene and C-MET mRNA,but decreases the Caspase3/7 activity in MCF-7 cells under hypoxia.Conclusion The pcDNA3-HIF-1? eukaryotic expression vector was successfully constructed,it not only has strong DNA binding and inducing activity,but also has anti-apoptotic effect in hypoxia MCF-7 cells.

Construction of recombinant plasmid PIRES2-HPV18-EGFP and its expression in HeLa cells / 第三军医大学学报

Objective To construct a recombinant plasmid for expressing the HPV18 E2 gene and develop some materials for HPV18 related disease.Methods The E2 gene of HPV18 was amplified by PCR from PBR322-HPV18 and cloned into PIRES2-EGFP.The sequence of cloned HPV18E2 was confirmed by restriction analysis and DNA sequencing,then transfected into HeLa cells.E2 gene was detected by RT-PCR.Results HPV18 E2 gene fragment was amplified by PCR and ligated to PIRES2-EGFP,then transfected into HeLa cells successfully.The expression of green fluorescence cells was observed by fluorescence microscopy.Specific band could be seen in transfected cells by RT-PCR.Conclusion Recombinant plasmid PIRES2-EGFP is effectively expressed after being transfected into HeLa cells in vitro.

Construction of pEGFP-VEGF recombinant plasmid and its expression in osteoblast / 重庆医科大学学报

Objective:To construct a vector containing human vascular endothelial growth factor,VEGF 121 (VEGF 121 ) with green fluorescence protein as the reporter gene,and then to observe its expression in osteoblast.Methods:VEGF 121 gene was amplified from foetus brain library by PCR,then ligated with pEGFP-C1 to form recombinant plasmid pEGFP-VEGF and transfected into osteoblast by liposome.The green fluorescence protein expression was observed by fluorescence microscopy and the VEGF expression was last detected by immunocytochemistry and ELISA.Results:VEGF 121 gene fragment was amplified by PCR and ligated to pEGFP-C1,then transfected into osteoblast successfully.The strong expression of green fluorecensce protein was observed by fluorecensce microscopy,and the expression of VEGF protein was detected by immunocytochemistry and ELISA.Conclusion:Recombinant plasmid pEGFP-VEGF is effectivly expressed after being transfected into osteoblast in vitro,which provides theoretical support for further study with its simple and reliable detecting methods.