The fused gene (PTH-TFN) of parathyroid hormone (PTH) gene and transferring N-terminal half-molecule (TFN) gene was amplified by multiple PCR and inserted into pPIC9 vector. The recombinant plasmid pPIC9-PTH-TFN was transformed into Pichia pastoris GS115 by PEG. After methanol induction, the target protein was expressed in fermentation supernatant at high level. The fused protein PTH-TFN with purity being higher than 95% was finally obtained after purification through two-step chromatography: SP Sepharose Fast Flow and Phenyl Sepharose Fast Flow. Western blot analysis and adenylate cyclase assay proved that the fused protein exhibited the bioactivity to stimulate cAMP synthesis and the ability to bind Fe3+ in the Fe3+ saturation study as the recombinant TFN did indicating that TFN could be used as the transcellar carrier of PTH.
Artificial Gene Fusion ; Cloning, Molecular ; Humans ; Parathyroid Hormone ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transferrin ; genetics

To construct the ss/HBsAg protein gene-engineering vaccine for developing the diagnosis and cure tumors in clinical medicine and promoting the growth in animal husbandry production. A pair of primers were designed according separately to the sequence of Somatostatin gene(S14) and HBsAg gene. Their gene fragments were separately amplified by using PCR and cloned, following sequencing, the DNA fragments were inserted into pBluescript vector. Then the ss/HBsAg chimera was constructed and was cloned into pPICZaA plasmid, and transformed into electroporated Pichia pastoris. High yield protein expression was obtained. Expressed protein was proved with high specificity and it's molecular weigh was about 28 KD identified by SDS-PAGE and Western blot.
Artificial Gene Fusion ; Cloning, Molecular ; Gene Expression ; Genetic Vectors ; Hepatitis B Surface Antigens ; genetics ; Pichia ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; Somatostatin ; genetics

OBJECTIVE: To construct nasopharyngeal carcinoma CNE-2 cell lines expressing stable fusion suicide gene CD/UPRT. UL49. METHOD: The plasmids of pcDNA3.1 (-)E6. BARF1p. CD/UPRT. UL49 was transfected into CNE-2 cells through lipofectamine, and the transfected CNE-2 cells were selected by G418 and prodrugs for getting the cells expressing fusion CD/UPRT. UL49 gene. The protein produced by the suicide gene was tested by Western-blotting in CNE-2 cells. RESULT: Suicide genes were expressed stably in CNE-2 cells. CONCLUSION We constructed nasopharyngeal carcinoma cell lines CNE-2 expressing stable suicide gene through lipofectamine.
Artificial Gene Fusion ; methods ; Carcinoma ; Cell Line, Tumor ; Genes, Transgenic, Suicide ; Humans ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; genetics

A fusion gene CTB-PROIN, in which Proinsulin gene was fused to the 3' end of CTB gene by a hinge peptide 'GPGP', was constructed and cloned into pET-30a(+) to obtain a prokaryotic expression vector pETCPI. Subsequently the recombinant plasmid pETCPI was transformed into E. coli stain BL21 (DE3). After induced by IPTG, the expression product was analyzed by sodium dodecyl sulphate-polyacrylamide gel (15%) electrophoresis (SDS-PAGE), and its result indicated that the recombinant protein CTB-PROIN was expressed and accumulated as inclusion bodies. The recombinant CTB-PROIN protein accumulated to the level of 25% of total bacterial proteins. After inclusion bodies was denaturalized and refolded in vitro, significant assembly of monomers had occurred, and the recombinant protein represented assembled pentamers. The results of western blotting analysis also demonstrated that the fusion protein could be recognized by the anti-CT and anti-insulin antibody, respectively. In addition, the result of the CTB-PROIN-GM1 binding assay, that the protein could bind to monosialoganglioside specifically, showed it possesed biological activity in vitro. These results provided the possibility of developing a cheaper and more efficient oral vaccine for type I diabetes using such constructs.
Artificial Gene Fusion ; Cholera Toxin ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; G(M1) Ganglioside ; metabolism ; Proinsulin ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

In the current work, the fusion gene including somatostatin (SS) and the hepatitis B surface antigen gene was cloned into a balanced lethal system plasmid (pYA3493), and then transformed into asd- attenuated Salmonella choleraesuis C500 strain, the positive transformant without antibiotic resistance gene was confirmed by restriction analysis and DNA sequencing, designated as pYA-SS. The expression and immunogenicity of fusion protein were detected by SDS-PAGE and Western blot analysis. These results show that the recombinant prokaryotic expression plasmid pYA-SS could express the SS fusion protein with good immunogenicity in C500 strain. In above all, this study could provide reliable materials to develop novel, good and safe vaccine in enhancing the growth of animals.
Animals ; Artificial Gene Fusion ; Cloning, Molecular ; Hepatitis B Surface Antigens ; genetics ; Humans ; Plasmids ; genetics ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Salmonella arizonae ; genetics ; metabolism ; Somatostatin ; biosynthesis ; genetics ; immunology

Coding sequences of BMP-2 and BMP-7 were amplified using PCR and ligated with a DNA sequence encoding a flexible peptide (Gly4Ser)5. The fusion gene was inserted into plasmid pIRESneo3. The expression level of BMP2/7 heterodimers in the transfected CHO-K1 cells was 230.75+/-13.34 ng/mL. Culture medium of stably tansfected clone pool was collected as conditional medium to treat osteoblast MC3T3 cells. Staining of Alkalin phosphatase and Alizarin red demonstrated that the conditional medium significantly promoted osteogenic differentiation to a higher extent than BMP-2 homodimers expressed in either CHO-K1 cells or E. coli. Transcriptional levels of Osteogenic phenotype-related molecular markers such as OC, ALP, Runx2 and Osx were increased (P<0.05), and BMP/Smad signal activities were significantly enhanced by BMP-2/7 heterodimers, comparing with BMP-2 homodimers (P<0.05). The results demonstrate that BMP-2/7 heterodimers expressed in CHO-K1 cells have potent ability to stimulate osteogenic differentiation.
3T3 Cells ; Animals ; Artificial Gene Fusion ; Bone Morphogenetic Protein 2 ; biosynthesis ; genetics ; Bone Morphogenetic Protein 7 ; biosynthesis ; genetics ; CHO Cells ; Cell Differentiation ; drug effects ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Dimerization ; Escherichia coli ; genetics ; metabolism ; Gene Fusion ; genetics ; Humans ; Mice ; Osteoblasts ; cytology ; Osteogenesis ; drug effects ; Transfection

Recently, cancer therapy with mutiple genes has been attached with great attention. However, at present there is no efficient tool to construct multiple-cistrons. The large sizes and the imbalance in expression of most traditional tools, such as ribosome entry sites (IRESes),greatly block their wide employment in the construction of multiple cistronic gene therapy vectors. The self-cleaving peptide 2A from foot-and-mouth disease virus (FMDV) has a very small size, and more importantly, high cleavage activity in artifical bicistron, which bring new hope for mutiple genes therapy stategy. In this article, the characteristics and cleavage activities of FMDV 2A will be elucidated,and we further outline its applications in cancer gene therapy.
Amino Acid Sequence ; Artificial Gene Fusion ; DNA, Recombinant ; Foot-and-Mouth Disease Virus ; genetics ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Neoplasms ; therapy ; Promoter Regions, Genetic ; RNA Splicing ; Transcription Factors

The onset, severity, and ultimate outcome of malaria infection are influenced by parasite-expressed virulence factors and individual host responses to these determinants. In both humans and mice, liver injury is involved after parasite entry, which persists until the erythrocyte stage after infection with the fatal strain Plasmodium falciparum (Pf). Hepatocyte growth factor (HGF) has strong anti-apoptotic effects in various kinds of cells, and also has diverse metabolic functions. In this work, Pf-subtilisin-like protease 2 (Pf-Sub2) 5'untranslated region (UTR) was analyzed and its transcriptional activity was estimated by luciferase expression. Fourteen TATA boxes were observed but only one Oct-1 and c-Myb were done. In addition, host HGF interaction with Pf-Sub2 was evaluated by co-transfection of HGF- and Pf-Sub2-cloned vector. Interestingly, -1,422/+12 UTR exhibited the strongest luciferase activity but -329 to +12 UTR did not exhibit luciferase activity. Moreover, as compared with the control of unexpressed HGF, the HGF protein suppressed luciferase expression driven by the 5'untranslated region of the Pf-Sub2 promoter. Taken together, it is suggested that HGF controls and interacts with the promoter region of the Pf-Sub2 gene.
*5' Untranslated Regions ; Artificial Gene Fusion ; Cell Line ; Genes, Reporter ; Hepatocyte Growth Factor/*metabolism ; Hepatocytes/parasitology ; *Host-Parasite Interactions ; Humans ; Luciferases/genetics/metabolism ; Plasmodium falciparum/*pathogenicity ; Protein Binding ; Subtilisins ; *Transcription, Genetic

To enhance the immuogenicity of DNA vaccines expressing the GP5 protein of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the tegument protein VP22 (encoded by VP22 gene) of Bovine Herpesvirus 1 (BHV-1), which has been demonstrated to exhibit the unusual protein transduction property, was fused to N-terminus of GP5 of DNA vaccine construct pCI-ORF5M, resulting in pCI-VP22-ORF5M expressing VP22-GP5 fusion protein. The expression of VP22-GP5 fusion protein was confirmed by both indirect immunofluorescence assay (IFA) and Western blot. To investigate its immunogenicity, BALB/c mice were immunized with the fusion expression plasmid pCI-VP22-ORF5M and non-fusion expression plasmid pCI-ORF5M, respectively. The GP5-specific ELISA antibodies, neutralizing antibodies and lymphocyte proliferative responses were evaluated at various time points after primary immunization. The results showed that GP5-specific ELISA antibodies, neutralizing antibodies, and lymphocyte proliferative responses induced by DNA vaccine pCI-VP22-ORF5M were higher significantly than those of DNA vaccine pCI-ORF5M, indicating that fusion expression with BHV-1 VP22 significantly enhances the immuogenicity of DNA vaccine expressing the PRRSV GP5 protein, and that this strategy may also be useful to develop more efficient DNA vaccines against other pathogens.
Animals ; Antigens, Viral ; genetics ; immunology ; Artificial Gene Fusion ; Female ; Mice ; Mice, Inbred BALB C ; Porcine Reproductive and Respiratory Syndrome ; prevention & control ; Random Allocation ; Vaccines, DNA ; genetics ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; Viral Structural Proteins ; genetics ; Viral Vaccines ; genetics ; immunology

The gene expressions of codeinone reductase (COR) and berberine bridge enzyme (BBE) in Papaver somniferum were blocked by RNA hairpin of RNA interference (RNAi). The complete sequences of COR and BBE genes were cloned by reverse transcription-polymerase chain reaction (RT-PCR), the results of homology comparison revealed that the cloned COR and BBE genes had high homology with the other gene family members reported in the GenBank. The target sequences of COR and BBE genes were screened in accordance with the design principle of RNAi, a 643 bp fusion gene was obtained by the method of overlapping PCR, then plant expression vector ihpRNA was constructed based on intermediate vector pHANNIBAL and plant expression vector pCEPSPS. With that 78 transgenic plants were obtained through Agrobacterium-mediated and 17 positive plants were screened by PCR, that could initially indicate that the target fragments of COR and BBE gene had been integrated into tobacco genome.
Artificial Gene Fusion ; Genetic Vectors ; NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases ; genetics ; Oxidoreductases, N-Demethylating ; genetics ; Papaver ; enzymology ; genetics ; Plants, Genetically Modified ; enzymology ; genetics ; RNA Interference ; RNA, Small Interfering ; Tobacco ; genetics ; Transformation, Genetic