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.

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

Overexpression of the mTERT gene by adenoviral vectors promotes the proliferation of neuronal stem cells in vitro and stimulates neurogenesis in the hippocampus of mice

We sought to construct the adenoviral vector carrying the gene encoding mouse telomerase reverse transcriptase（mTERT）,as well as detect its expression and effect on the proliferation of neuronal stem cells.mTERT was am-plified by RT-PCR and then the eukaryotic expression vector of pDC-EGFP-TERT was constructed.After DNA sequence analysis,we detected that there were 293 cells transfected with pDC-EGFP-TERT and helper adenovirus plasmid pBHG lox ΔE1,and three Cre using Lipofectamine 2000 mediation,named Ad-mTERT-GFP,to pack-age adenoviral particles.The Ad-mTERT-GFP was used to infect neuronal stem cells and then the expression and activity of mTERT were detected.In addition,Bromodeoxyuridine labeling test identified the impact of mTERT overexpression on proliferation of neuronal stem cells.The recombinant adenoviral vector confirmed that mTERT was successfully constructed.Overexpression of mTERT stimulated the proliferation of neuronal stem cells both in vitro and in vivo.mTERT overexpression via adenoviral vector carrying mTERT cDNA upregulated the ability of proliferation in neuronal stem cells.

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 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 and Identification of a Human Liver Specific MicroRNA Eukaryotic Expression Vector

MiR-122 is one of the non-coding RNAs which showed its effects on the lipo-metablism, virus infection and HCC forming through regulation of liver gene expression. Its eukaryotic expression vector was constructed by using pSuper which was widely applied in the siRNA expression. The precursor of human miR-122 gene was amplified by polymerase chain reaction （PCR） from the human genomic DNA. The positive clones were screened by PCR and restriction enzyme digestion. The new expression vector of miR-122 was named pHsa-m122. PHsa-m122 and its controls were transfected to HepG2 cells. The miR-122 expression activity was evaluated by GFP122i sensor reporter plasmid through fluorescence detection and Western blot. It was shown that the fluorescence intensity of GFP122si and pHsa-m122 co-transfection group was weaker than that of the controls, so the functional activity of expressed miR-122 was detected. When HepG2 cells were co-transfected with HBV1.3 and pHsa-m122 plasmids, the results showed miR-122 may down-regulate the gene expression of HBV. The human liver specific microRNA eukaryotic expression vector of miR-122 was constructed successfully, which may facilitate further study of its function in the development of liver virus infection diseases and HCC. Cellular ＆ Molecular Immunology.

Stable Expression of Antibacterial Peptide CecropinB in Dairy Goat Mammary Gland Epithelial Cells

The paper aimed at constructing the eukaryotic expression vector of CecropinB and transfecting it into the goat mammary epithelial cell line. The recombinant plasmid inserted with CecropinB was transfected into the goat mammary epithelial cell by liposome Tfx^M-20. By screening with G418, the stable transfected goat mammary epithelial cell line was established and the transcription and expression of CecropinB were identified by RT-PCR and agarose diffusion experiment, respectively. Results showed that eukaryotic expression vector pECFP-B was constructed successfully. Stable transfected goat mammary epithelial cell line was established and the CecropinB protein was expressed successfully. It provides the solid foundation for further experimental studies on the function of CecropinB.

Construction of the pIRES2-ZsGreen1 eukaryotic expression vector of factor Ⅸ gene and expression in HEK-293 cells

Objective To construct p IRES2-ZsG reen1/FⅨexpression vector,using the pcDNA/FⅨplasmid containing FⅨcDNA as template,and expressing in HEK-293cells.Methods The total ORF of FⅨgene was amlified from pcDNA/FⅨplasmid,then the amplified fragment was clonded into the p IRES2-ZsG reen1 vector using

The Effect of Glycation of CD59 on Complement-Mediated Cytolysis

Vascular proliferation is one of the major causes for morbidity and mortality in diabetes. However, the cellular and molecular mechanisms that link hyperglycemia to this complication remain unclear. In present study, we demonstrated by site-directed mutagenesis that mutated CD59 was more susceptible to glycation-inactivation for hyperglycemia. Mutated and wild-type CD59s were stably expressed in Chinese hamster ovary cells using the pALTER-MAX mammalian expression vector. Western blot, FACS and immunological fluorescence were conducted to confirm that CD59s were tethered to the plasma membrane. Compared to wild-type CD59, human CD59 mutants led to a significant increase in dye release assay. These results indicate that there may be some mutations of CD59 in diabetes population and the mutated CD59, which is more likely to be of glycation than the wild-type, may help to explain the distinct propensity of diabetes subjects to develop vascular proliferation complications.