The Construction and Identification of Eukaryotic Expression Vector pEGFP-N1-hTERT

[Objective] The aim of this study is to construct eukaryotic expression vector pEGFP-N1-hTERT and observe its expression in eukaryotic cells.[Method]The eukaryotic expression vector pEGFP-N1-hTERT was constructed with pC1-neo-hTERT and pEGFP-N1 plasmids,and the accuracy of human telomerase reverse transcriptase(hTERT)gene fragment was confirmed by double enzyme digestion and DNA sequencing analysis.After transfecting pEGFP-N1-hTERT into rat fetal neural stem cells(NSCs),the protein localization of human telomerase reverse transcriptase were indirectly observed through green fluorescent protein in the cells,and the correctness of constructed pEGFP-N1-hTERT was certificated by RT-PCR and Western Blot analysis.[Result]The eukaryotic expression vector pEGFP-N1-hTERT had correct structure and could express in eukaryotic cells.[Conclusion]This study laid a foundation for the establishment of immortalized NSCs line in rats.

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 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.

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.

Gene Cloning of Murine α-Fetoprotein Gene and Construction of Its Eukaryotic Expression Vector and Expression in CHO Cells

To clone the murine α fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1 6 cells, and then the murine α fetoprotein gene was amplified by RT PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.

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 bicistronic green fluorescent protein labeled pSELECT GFPzeo human bone morphogenetic protein 2 eukaryotic expression vector

Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase

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.

Construction of Plant-based Expression Vector of Polyphosphate Kinase Gene

[Objective] The aim was to construct the fusion expression vector of polyphosphate kinase（PPK） and green fluorescent protein（GFP） genes.[Method] In this study,the primers were designed based on PPK gene sequence（L03719） of E.coli DH5α in Genbank.Genomic DNA of E.coli DH 5α was extracted as template for the amplification of PPK gene by PCR method.By using In-Fusion@ HD Cloning Kit,the PPK gene was directionally cloned into NcoI site of the pCAMBIA1302 vector.[Result] Sequencing results showed that the 2.0 kb long fragment of PPK gene was inserted into the plant-based expression vector pCAMBIA1302 in front of GFP gene.[Conclusion] The fusion expression vector of PPK and GFP genes were successfully constructed.

Construction of Plant Expression Vector for Recombinant Human Epidermal Growth Factor (hEGF)

ObjectiveThis study aimed to construct plant expression vector for recombinant human epidermal growth factor （hEGF） and further to provide a basis for the expression of hEGF in peanut hairy root system. MethodAccording to the hEGF sequence in GenBank, hEGF was synthesized artificially; subsequently, hEGF gene was ligated with green fluorescent protein （GFP） gene, and their ligation product was then amplified with primers flanked with corresponding endonuclease cleavage sites, followed by double digestion by Sal I and EcoR I of the amplified products; next, pRI 101 AN DNA was extracted and digested by both Sal I and EcoR I; susequently, the digestion products of hEGF and GFP ligation fragment by Sal I and EcoR I and the digestion products of pRI 101 AN plasmid DNA by Sal I and EcoR I were ligated, and their ligation product was transformed into Escherichia coli XL10-Gold, followed by extraction of DNA from the recombinants exhibiting green fluorescence, which was then identified by enzymatic digestion and PCR, and the verified recombinant plasmid DNA was named pBZG101. ResultHuman epidermal growth factor gene （hEGF） and green fluorescent protein gene （GFP） were successfully ligated, and their ligation fragment was successfully ligated to pRI 101 AN DNA, finally with the acquirement of the plant expression vector for recombinant human epidermal growth factor-（pBZG101）. ConclusionThe plant expression vector for recombinant human epidermal growth factor-（pBZG101）- was successfully constructed in this study.

Construction of Plant Virus Expression Vector pClYVV/CP/W and Expression of GFP

[ Objective] The study was to report the construction of plant virus expression vector pCIYVV/CP/W and the expression of green fluorescent protein（GFP） with pCIYVV/CP/W, and to develop effective plat virus vector for plant bioreactor to produce useful protein. [ Method] A section of multiple cloning sites among NIb/CP genes in pCIYVV genome and deoxyribonucleotide polylinker of cleavage recognition sequence containing viral protease Nla were cloned with infectivity full-length cDNA of clover yellow vein virus （CIYVV）, and pCIYVV/CP/W vector was constructed, GFP gene was inserted into pCIyVV/CP/W to construct the pCIYVV/CP/W/GFP vector. The transcription situation of recombinant virus clone was detected by RT-PCR, and targeted gene products expressed by recombinant virus clone were detected with western blot （WB）. [Result] The broad bean seedling inoculated with pCIYVV/CP/W/GFP expressed the same symptom as wild type CIYVV, morbidity was of 100%, the result showed that recombinant virus clone pCIYVV/CP/W/GFP didn＇t suppress, insertion of foreign gene didn＇t destroy the open reading frame of pCIYVV/CP/W. Foreign gene can keep living in F, progeny virus genorne steadily, recombinant virus clone pCIYVV/CP/W/GFP could steadily express GFP in progeny virus at least.[ Conclusion] The useful plant virus vector was provided for useful protein expressing.

Expression Vector Construction and Genetic Transformation of <i>Rosa rugosa β</i>-l,3-Glucanase Gene (<i>RrGlu</i>)

In order to lay a foundation for researching the function of Rosa rugose (R. rugosa) RrGlu gene, the RrGlu gene was amplified from the styles of R. rugosa “Tanghong”, a gene expression vector named PBI121-RrGlu was constructed and the vector was introduced into tobacco with the agrobacterium-mediated method. PCR results showed that the RrGlu gene was integrated into the tobacco genome.

Construction of Expression Vector for Porcine Gastrin-releasing Peptide Fusion Protein

[Objectives]This study was conducted to obtain porcine gastrin-releasing peptide(GRP)fusion protein.[Methods]The constructed pET32a(+)-GRP plasmid was transformed into Escherichia coli BL21(DE3)competent cells to obtain the pET32a(+)-GRP-BL21(DE3)fusion protein expression strain,which was induced with 0.5 mM IPTG at 25℃and 150 r/min for 12 h,and the His-tagged GRP fusion protein was detected by SDS-Page gel electrophoresis and Western Blot.[Results]After optimizing the IPTG-induced expression conditions,it was confirmed that the porcine GRP fusion protein was obtained,and the porcine GRP fusion protein was soluble,stable and highly active.[Conclusions]This study lays a foundation for the subsequent preparation of anti-pig GRP antibodies.

Construction of Smac gene-carrying and human uroplakin Ib promoter-Regulated Genetic Vector and its Expression

Objective： To construct an eukaryotic expression vector that contains Smac gene, which is regulated by human Uroplakin Ib （UpIb） promoter. Methods： For the directionality of Smac expression in the transitional cell carcinoma of bladder, internal CMV and T7 promoter sequences in eukaryotic expression vector pcDNA3.1-Smac were replaced with UpIb promoter to construct a new plasmid. The plasmid DNA was identified by gel electrophoresis after being double digested at respective sites, and then the sequence was analyzed. The expression of Smac mRNA and protein in BIU87 cell line were detected after the transfection by using the newly constructed vector. Results： The Smac gene-carrying and UpIb promoter-regulated eukaryotic expression vector pcDNA3-UpIb-promoter-Smac was successfully constructed. The expression of Smac mRNA was approximately increased by 2.1 times and the expression of Smac protein was increased in about 71% BIU87 cells. Conclusion： The new vector can be effectively expressed in bladder cancer cells and be of great significance for bladder cancer-targeted gene therapy.

Construction of a HERG mutant L539fs/47-*558W pEGFP vector and the expression of the fusion protein in HEK293 cells

Objective： To construct a human ether-a-go-go-related gene （HERG） nonsense mutant L539fs/47-558W into the autonomously fluorescent, eukaryotic expression vector pEGFP-C2, and to verify expression of the reconstruct in human embryonic kidney-293 （HEK293） cells. Methods： The mutational fragment was subcloned into pEGFP-C2-HERG by double digestion of Sbf I, Eco91 1 and rejoining of T4 ligase. After verification, the recombinant pEGFP-C2-L539fs/47-558W and pEGFP-C2-HERG were respectively transfected into HEK293 cells for 48 h by the Lipofect method to observe the expression location of the fusion protein by laser confocal imaging scanning in vivo. pcDNA3 -L539fs/47-＊558W and pcDNA3-HERG were transfected to observe the expression location of the HERG protein by immunofluoresceoce. The mutant protein size was determined by Western blotting. Results： The about 1 kb-sized mutation region cDNA fragment from pcDNA3-L539fs/47-＊558Wand the about 7.2 kb-sized target vector fragment from pcDNA3-HERG were ligated after purification and gel recovery pEGFP-C2-L539fs/47＊-558W, approximately 8.2 kb, was demonstrated successfully been constructed under agarose gel electrophoresis and further sequencing. Laser confocal imaging showed that pEGFP-C2-HERG was mainly expressed in the membrane, whereas truncated mutant-type HERG in the pEGFP-C2 vector was partially located in the cytoplasm, the others were transported to the cell membrane in living HEK293 cells. The same as the immunofluoresceoce results after transfection of pcDNA3-HERG and pcDNA3-L539fs/47-558W. Wild-type HERG-GFP fusion protein expressed 160 and 180 kDa bands. The mutant and mutant-GFP fusion proteins were 70 and 100 kDa, respectively. Conclusion： pEGFP-C2-L539fs/47-＊558W was successfully constructed by double digestion method GFP had no effect on its protein expression and trafficking in HEK293 cells, which laid a foundation for the further study on L539fs/47-＊558W