Eukaryotic expression, purification and activity characterization of human soluble DSG2 extracellular domain protein

Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.Methods:The DSG2 extracellular domain fragment gene(DSG2ex),was amplified by PCR,and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1.The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein.The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA.Results:The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed.The highest protein expression level was obtained with 293T cells after 96 h of transfection.The relative molecular mass of the purified product was between 100 and 130 kDa was estimated by SDS-PAGE,which was consistent with the expectation.The yield of the purified protein reached 0.8 mg/ml with a purity over 90%.The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting.Moreover,the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein(HAdV-55).Conclusion:A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established,and biologically active DSG2 extracellular domain protein was purified,which laid the foundation for the later study of its protein function and anti-adenovirus drugs.

Construction and Preliminary Identification of Eukaryotic Expression Vector of Cryptosporidium parvum miR-2980

[Objective] This study aimed to construct and preliminarily identify the eu- karyotic expression vector of Cryptosporidium parvum miR-2980. [Method] The cp-miR- 2980 precursor was amplified from C. parvum genomic DNA and cloned into pMD18- T vector. The amplified precursor was then subcloned into pVAX I vector and identi- fied with restriction endonuclease digestion and sequencing. The recombinant plasmid pVAX-miR2980 was transfected into HCT-8 cells. Total RNA was extracted and the expression of cp-miR-2980 was evaluated by RT-PCR detection. [Result] The results showed that the recombinant eukaryotic expression vector pVAX-miR2980 was suc- cessfully constructed, which can express cp-miR-2980 in HCT-8 cell. [Conclusion] This study laid the foundation for further exploring the biological function of cp-miR-2980.

Construction of Eukaryotic Expression Vector with Partial Encoding Sequence of Actin from Cryptosporidium andersoni

[Objective] To clone the actin gene of Cryptosporidium andersoni, and to study its eukaryotic expression in Hela cells. [Methed] Specific primers were designed for the partial encoding sequence of actin, which were obtained by screening the T7 phage display library of Cryptosporidium andersoni, and the actin gene CA42 was amplified by PCR. Recombinant eukaryotic expression plasmid pVAX1-CA42 was constructed and transfected to Hela cells with lipofection strategy. Indirect im- munofluorescence staining, SDS-PAGE and Western blotting analysis were used to detect the expression of recombinant protein in Hela cells. [Result] CA42 protein was successfully expressed in Hela cells, and the expression products had reactogenicity. [Conclusion] The partial encoding sequence of actin from Cryptosporidium andersoni has been successfully cloned, and it can be stably expressed in Hela Cells

Establishment of BHK-21 Stable Cell Lines Expressing T7 RNAP and GFP

[Objective] The aim was to establish the BHK-21 stable cell lines expressing T7 RNAP and GFP.[Method]T7 RNAP gene was amplified from E.coli BL21（DE3）and inserted into FG12 vector.The lenti-virus recombinant plasmid FG12-T7 RNAP plasmid was obtained via identification with double enzymes digestion and gene sequencing.The transient expressed T7 RNAP protein was determined by WB in 293T cells transfected with FG12-T7 RNAP plasmid.The recombinant FG12-RNAP lenti-virus was packaged up by transfecting the 293 cells with the recombinant vector FG12-RNAP and the helper plasmids via lipofectamine-2000,which was then used to infect BHK-21 cells.The positive cell clones were obtained after continuous screening by GFP.The expression of T7 RNAP gene was confirmed by Western blot.[Result]The cell line stably expressing the T7 RNAP gene was established by Western blot.[Conclusion]T7 RNAP gene could be stably expressed in eukaryotic cells and it provided a good platform to rescue RNA virus in vivo.

Construction of Eukaryotic Expression Vector for Pig Ghrelin Gene

[Objective] This study aimed to investigate the functions of transgenic growth related gene in pig growth. [Method] A pair of primers containing Nhe I and Hind Ⅲ restriction sites were designed by referring to the pig Ghrelin mRNA sequence published in Genbank. Total RNA was extracted from the small intestine tissue of 13/17 Robertson translocation heterozygous pig, and then was purified and used as the template in later RT-PCR reaction to amplify the full-length pig Ghrelin gene. The correct pig Ghrelin gene fragment was cloned into the pMD19-T simple vector for sequencing analysis. The obtained full-length cDNA of pig Ghrelin gene fragment was digested with both Nhe I and Hind Ⅲ, and then was linked into the eukaryotic expression vector pEGFP-N1 to obtain the recombinant plasmid pEGFPGhrelin. The recombinant plasmid was transected into the fibroblast cells to detect the fluorescence labeled gene expression. [Result] The nucleotide sequence extracted from 13/17 Robertson translocation heterozygous pig was the same as expected; and the eukaryotic expression vector pEGFP-Ghrelin was successfully constructed. [Conclusion] The eukaryotic expression vector constructed in this study can be further used in research on transgenic pigs, but also lays foundation for research on the regulatory mechanism of Ghrelin gene.

Cloning and construction of sense and antisense eukaryotic expression vector of human Pin1

Objective： To clone and construct eukaryotic expressing vectors of sense and antisense human Pin1 （hPinl） genes. Methods： Total RNA was extracted from MG-63 cells, then the hPinl cDNA was amplified by RT-PCR. The same time the sense and antisense hPinl genes were formed by binding BamH Ⅰ and Hind Ⅲ in cis and trans-directions. At the end they were cloned into the eukaryotic expressing vector pIRES2-EGFP in cis and trans directions using DNA recombinant technology. The recombinant vectors were further identified by digestion of BamHⅠ and Hind Ⅲ. Results： The results of sequencing showed that the orientation of the ligations and the reading frame were correct. After digested by BamH Ⅰ and Hind Ⅲ, two fragments exhibiting 5.3 kb and 0.99 kb were formed in sense and antisense eukaryotic expressing vectors. Electrophoretic results were completely coincident with theoretical calculation. Conclusion： Human Pin1 sense and antisense genes were successfully cloned and eukaryotic expressing vectors were successfully constructed.

Construction of PR domain eukaryotic expression vector and its inhibitory effect on esophageal cancer cells

Objective：PR domain is responsible for the tumor suppressing activity of RIZ1.The study aimed to construct human PR domain eukaryotic expression vectors,transfect human esophageal cancer cells （TE13）,and evaluate the anticancer activity of PR domain on human esophageal cancer TE13 cells.Methods：First,mRNA was extracted from human esophageal cancer tissue by RT-PCR,then reversetranscribed to cDNA.After amplifying from the DNA template,PR domain was linked to T vector.Second,after extraction,PR domain was cut using enzyme and linked to pcDNA3.1（＋）.Then,the plasmid was transfered to Trans1-T1 phage resistant competent cells,following by extracting the ultrapure plasmid,and transfecting into TE13 cells.In the end,the protein expression of pcDNA3.1（＋）/PR domain in TE13 was detected by Western blot,and the apoptosis of TE 13 by technique of flow cytometry.Results：More than 5,000 bp purposed band of pcDNA3.1（＋）/PR domain plasmid was found by agarose gel electrophoresis.After transfection,the PR domain （molecular weight of about 28 Da） was found only in 3,4 and 5 groups by Western blot.Flow cytometry assay showed apoptosis in experimental group was significantly more than that in the control group （P＜0.05）.Conclusions：The PR domain eukaryotic expression vector was constructed successfully.The protein of the PR domain could be expressed in esophageal cancer TE13 cells firmly after transfection,and a single PR domain could promote apoptosis of TE13 cells.

Construction of human eukaryotic expression plasmid vascular endothelial growth factor 165 and its expression in transfected vascular smooth muscles

BACKGROUND: The highly specific vascular endothelialgrowth factor (VEGF) induces the growth of vascular en-dothelial cell. This study was to construct the eukaryoticexpression plasmid of vascular endothelial growth factorl65(VEGF165) and observe its expression in vascular smoothmuscles (VSMCs).METHODS: The primers were designed and synthesizedaccording to the gene sequences of human VEGF165. TheVEGF165 gene was obtained from umbilic artery tissue bythe method of RT-PCR, then it was cloned to eukaryoticexpression plasmid pBudCE4.1 by recombination strategy.The eukaryotic expression plasmid named pBudCE4.1/VEGF165 was identified by restriction enzyme digestion,and was sequenced. The pBudCE4.1/VEGF165 was trans-fected into VSMCs by using lipofection. The VEGF165 ex-pression of mRNA and protein was detected by RT-PCRand Western blot respectively.RESULTS: VEGF165 was shown about 576bp by RT-PCR.Sequencing revealed the amplified VEGF165 gene was iden-tical with that in the GeneBank. Restrictive enzyme (HindBam HI) digestion analysis showed that recombinantexpression plasmid pBudCE4. l/tVEGF165 had been con-structed successfully. The expression of VEGF165 at mRNAand protein levels in the transformed VSMCs had beendemonstrated by RT-PCR and Western blot.CONCLUSIONS: The recombinant eukaryotic expressionplasmid pBudCE4.1/VEGF165 has been successfully con-structed and expressed in transformed VSMCs. The presentstudy has laid a foundation for VEGF165 gene therapy ofvascular stenosis in the transplant organ.

CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR WITH GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR GENE

Objective: To construct the eukaryotic expression vector that express human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene for making highly express in mammalian cells. Methods: Extract totally RNA from the induced human fetal lung (HFL) cell line. HGM-CSF cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR), and then directionally subcloned into the HindIII and EcoRI site on the pcDNA3.1 plasmid, which was controlled by the CMV promoter, to form the recombinant expressing vector pcDNA3.1-GM-CSF. Results: The PCR amplification was identified and the sequence was analyzed, the results showed that hGM-CSF was properly inserted into the vector and the sequence was correct.

Construction of human VEGF165 gene eukaryotic expression plasmid and its effect on proliferation of vascular endothelial cells

After organ transplantation, rapid repair of injured vascular endothelial cell (VEC) is a key to prevent graft chronic dysfunction besides control of immunological rejection. Many studies have confirmed that vascular endothelial growth factor 165 (VEGF165) could accelerate the repair of VEC injury, decrease thrombosis and thrombotic occlusion, and inhibit hyperplasia of the intima. This study was designed to construct eukaryotic expression plasmid pBudCE4.1/VEGF165, and observe its effect on the prolife ration of VEC. METHODS:The VEGF165 gene cloned from human heart tissue by RT-PCR was cloned into eukaryotic expression plasmid pBudCE4.1. The recombinant expression plasmid pBudCE4.1/VEGF165 was identified by restriction enzyme (Hind III and BamH I) digestion analysis, and was sequenced. The pBudCE4.1/VEGF165 was introduced into VEC through lipofection transfection. The VEGF165 mRNA expression by Northern blot and VEGF165 protein expression was detected by immunocytochemical staining. The effect of expression protein on VEC proliferation was detected by flow cytometry. RESULTS:The RT-PCR product of the VEGF165 gene was about 576bp. Sequencing analysis revealed that the sequence of the amplified VEGF165 gene was identical with that in GenBank. Restrictive enzyme digestion analysis showed that recombinant expression plasmid pBudCE4.1/ tVEGF165 had been constructed successfully. The expression of VEGF165 at mRNA and protein levels in the transformed VSMCs had been demonstrated by Northern blot and immunocytochemical staining respectively. The expressed product of VEGF165 could notably accelerate the proliferation of VECs. CONCLUSIONS:pBudCE4.1/VEGF165 is successfully cons- tructed and is expressed in VECs. Expressed VEGF165 can accelerate the VEC proliferation. The present study has laid a foundation for potential use of VEGF165 gene transfection to prevent and treat vascular stenosis in the transplanted organ.

Eukaryotic Expression of Human Arresten Gene and Its Effect on the Proliferation of Vascular Smooth Muscle Cells

The eukaryotic expression of human arresten gene and its effect on the proliferation of in vitro cultured vascular smooth cells （VSMCs） in vitro were investigated. COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome. Forty-eight h after transfection, reverse transcription-polymerase chain reaction （RT-PCR） was used to detect the expression of arresten mRNA in the cells, while Western blot assay was applied to detect the expression of arresten protein in concentrated supernatant. Primary VSMCs from thoracic aorta of male Sprague-Dawley rats were cultured using the tissue explant method, and identified by immunohistochemical staining with a smooth muscle-specific anti-α- actin monoclonal antibody before serial subcuhivation. VSMCs were then co-cultured with the concentrated supernatant and their proliferation was detected using Cell Counting Kit-8 （CCK-8） in vitro. The results showed that RT-PCR revealed that the genome of arresten-transfected cells contained a 449 bp specific fragment of arresten gene, suggesting the successful transfection. Success- ful protein expression in supernatants was confirmed by Western blot. CCK-8 assay showed that the proliferation of VSMCs were inhibited significantly by arresten protein as compared with control cells （F=40. 154, P〈0.01）. It was concluded that arresten protein expressed in eukaryotic cells can inhibit proliferation of VSMCs effectively in vitro, which would provide possibility to the animal experiments.

CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR FOR HUMAN CCL21 AND CHARACTERIZATION OF ITS CHEMOTACTIC ACTIVITY

Objective： To obtain recombinant human CCL21 with biological activity from eukary0tic expression system for further use in cancer gene therapy. Methods： A fragment of human CCL21 gene was obtained from pSK-hCCL21 plasmid digested by Xho I and BamH I, inserted into the responding sites of eukaryotic expression vector pVAX1, and then transfected into COS-7 cells by electroporation method. The expression of hCCL21 protein was detected by western blotting analysis. The in vitro chemotaxis assay was used to test the chemotactic function of the expression product to lymphocytes. Results： Human CCL21 protein was expressed by transfected COS-7 cells with recombinant plasmid containing hCCL21 gene, and was verified by western blotting. The in vitro chemotaxis assay demonstrated that human CCL21 protein had a potent chemotactic function to lymphocytes. Conclusion： Human CCL21 was successfully and transiently expressed in eukaryotic cells, which lays some foundation for the study of CCL21 gene therapy in murine tumor models.

Eukaryotic expression and biological activity analysis of neuroprotective peptide [Gly14]-Humanin

Objective To investigate the expression of neuroprotective peptide [Gly14]-Humanin (HNG) in eukaryotic cells by gene engineering technique and analyze its biological activity. Methods By means of asymmetrical primer/template,double stranded cDNA of HNG with FLAG in its C-terminal was obtained,which was cloned into the plasmid pcDNA3.1(-),and the resultant recombinant vector pcDNA3.1(-)/HNG-FLAG was transfected into PC12 cells. At the same time,the recombinant vector pcDNA3.1(-)/EGFP was transfected to control the efficiency of transfection. The expression of HNG in the cells was determined by immunocytochemistry. In order to analyze the biological activity of the expressed HNG,25μM Aβ25-35 peptide was added to the culture medium of the transfected cells for 24h,then cell morphology,MTT assay and Hoechst 33258 staining were observed. Results The eukaryotic expression vector of pcDNA3.1(-)/HNG-FLAG was identified by enzyme digestion and sequencing. HNG was highly expressed in PC12 cells. After exposure of PC12 cells to 25μM Aβ25-35 for 24h,cell viability decreased to (65.8±5.3)%,and the dystrophic changes of neuritis and nuclei condensation were obvious. When cells were pre-transfected with pcDNA3.1(-)/HNG-FLAG,Aβ25-35-induced cell death and morphological changes of cells and nuclei were suppressed. In contrast,pre-transfected with empty vector did not protect cells from Aβ25-35-induced toxicity. Conclusion The eukaryotic expression vector for FLAG-tagged HNG was successfully constructed and expressed in PC12 cells. Expressed HNG has biological activity.

Science Letters:Construction of a eukaryotic expression plasmid of Humanin

Objective:To construct a eukaryotic expression plasmid pcDNA3.1(-)-Humanin.Methods:The recombinant plasmidpGEMEX-1-Humanin was digested with restriction endonucleases BamH Ⅰ and Hind Ⅲ and the Humanin gene fragments,about100 bp length,were obtained.Then the Humanin gene fragments were inserted into eukaryotic expression vector pcDNA3.1(-)andthe recombinant plasmids pcDNA3.1(-)-Humanin were identified by sequencing.Results:Recombinant plasmid DNA success-fully produced a band which had the same size as that of the thimauin positive control.The sequence of recombinant plasmidsaccorded with the Humnain gene sequence.Conclusions:A eukaryotic expression plasmid of Humanin was successfully con-structed.

Construction of Eukaryotic Expression Plasmid of bFGF Gene in Rats and Its Expression in Tenocytes

The bFGF plays an important role in embryonic development of tendons and ligaments and in the healing of injuried tendons and ligaments. The eukaryotic expression plasmid of rat basic fibroblast growth factor （bFGF） gene was constructed in order to further investigate the bFGF function in molecular regulatory mechanism in the repair of tendons and ligaments and to provide the foundation for the clinical application. The cDNA fragments of bFGF were cloned from the skin of rats by RT-PCR, and recombinated to the pMD18-T vector. The cDNA encoding bFGF was cloned from the pMD18-T vector by RT-PCR, digested with restriction enzyme EcoR Ⅰ, Pst Ⅰ and bound to eukaryotic expression plasmid plRES2-EGFP to construct eukaryotic expression plasmid plRES2-EGFP-bFGF. The plRES2-EGFP-bFGF was transfected into the tenocytes by lipid-mediated ransfection technique. MTT test was used to detect the biological activity of bFGF in supernatants after the transfection. The expression of type Ⅰ and Ⅲ collagen genes was detected by using RT-PCR. It was verified that the plRES2-EGFP-bFGF was successfully constructed, and its transfection into tenocytes could significantly enhance the biological activity of bFGF, and increase the expression of type Ⅰ and Ⅲ collagen mRNA, suggesting that plRES2-EGFP-mediated bFGF gene therapy was beneficial to the repair of tendons and ligaments.

Establishment of a Functional Cell Line Expressing both Subunits of H1a and H2c of Human Hepatocyte Surface Molecule ASGPR

To better understand the effect of a new split variant of human asialoglycoprotein receptor (ASGPR H1b) on ASGPR ligands’ binding ability, we established a functional cell line which expresses ASGPR.The full lengths of ASGPRH1a and H2c fragments from human liver were amplified by reverse transcript PCR (RT-PCR) and inserted into eukaryotic expression vector pIRES2EFP, pCDNA3.1 (Zeo+) respectively.The recombinants were cotransfected into HeLa cells.After selection by using Neocin and Zeocin, a stably transfected cell line was established, which was designated 4-1-6.The transcription and expression of ASGPRH1a and H2c in 4-1-6 were confirmed by RT-PCR, Western blotting and immunofluorescence.The endocytosis function of the artificial "ASGPR" on the surface of 4-1-6 was tested by FACS.It was found that the cell line 4-1-6 could bind ASGPR natural ligand molecular asialo-orosomucoid (ASOR).After the eukaryotic plasmid H1b/pCDNA3.1 (neo) was transfected into cell line 4-1-6, H1b did not down-regulate the ligand binding ability of ASGPR.The eukaryotic expression plasmid H1b/pcDNA3.1 (neo) and H2c/pcDNA3.1 (neo) were co-transfected transiently into Hela cell.Neither single H1b nor H1b and H2c could bind ASOR.In conclusion, a functional cell line of human asialoglycoprotein receptor (ASGPR) which expresses both H1a and H2c stably was established.The new split variant H1b has no effect on ASGPR binding to ASOR.ASGPRH1b alone can’t bind to ASOR, it yet can’t form functional complex with ASGPRH2c.

Construction and Expression of Eukaryotic Expression Vector of Mature Polypeptide of Duck Interferon Alpha Gene

To study biological activities of Duck Interferon Alpha （DuIFN-α） and prepare antivirus medicine, the eukaryotic expression vector of mature polypeptide of Duck Interferon Alpha （mDuIFN-α） gene was constructed and expressed in insect cell. By means of PCR technique, the mDuIFN-α gene was cloned from pMD-18-duIFN-α recombinant_ The gene was then inserted to pGEM-T vector and identified by restriction endonuclease analysis and sequencing The mDuIFN-α gene was ligated with the eukaryotic expression vector pMelBacA. then transfected into Sf9 cell line. Recombinant polypeptide was effectively expressed in insect cell and its molecular weight was 34 ku.