Human nerve growth factor (NGF) is a nerve cell growth regulation factor, which can provide nutrition for the neurons and promote the neurites outgrowth. In order to produce large-scale recombinant human nerve growth factor (rh-beta-NGF), we constructed a plasmid vector, which can stably express the rh-beta-NGF in the HEK293 cell lines. First, the plasmid of pCMV-beta-NGF-IRES-dhfr was constructed and transformed into HEK293 cells. Then MTX pressurized filter and limiting dilution methods were used to obtain monoclonal HEK293 cell lines. After stepwise reducing serum in culture media, the cells eventually adapted to serum-free medium and secreted rh-beta-NGF. SDS-PAGE analysis revealed that the expression product owned a molecular weight of about 13 kDa and a purity of more than 50%. The peptide mapping sequencing analysis demonstrated the sequences of rh-beta-NGF matched with the theoretical ones. Later we purified this protein by ion exchange and molecular sieve chromatograph. Finally, our experimental results exhibited that the recombinant cell lines can stably express rh-beta-NGF with a high efficiency of more than 20 pg/cell x day. In addition, this protein could successfully induce differentiation of PC12 cells. In summary, our recombinant HEK293 cells can express bio-active rh-beta-NGF with great efficiency and stability, which supply a valid basis to large-scale production of rh-beta-NGF.
Cell Differentiation ; Genetic Vectors ; HEK293 Cells ; Humans ; Nerve Growth Factor ; biosynthesis ; Plasmids ; Recombinant Proteins ; biosynthesis

The glucoamylase gene (glaA) of Aspergillus niger CICIM F0410 was cloned, sequenced and expressed. The integrated plasmid pBC-Hygro-glaA carrying the glaA was constructed and transformed into A. niger F0410. Transformants with multiple copies of glaA integrated in the chromosome were selected by 150 microg/mL hygromycin B and identified by real-time PCR. Two to three multiples of glaA in the chromosome were found to be optimal for higher expression of glucoamylase. Shake-flask fermentation under optimal conditions showed that glucoamylase secreted by the transformant GB0506 was 17.5% higher than parental strain F0410 at the end of fermentation. In conclusion, increasing copy number of glaA by chromosomal integration significantly improves the yield of glucoamylase in the industrial strain of A. niger.
Aspergillus niger ; genetics ; Cloning, Molecular ; Glucan 1,4-alpha-Glucosidase ; biosynthesis ; genetics ; Plasmids ; Recombinant Proteins ; biosynthesis

Radiotherapy is a treatment for cancer with undesired by-effects. In order to develop a new radiation protective agent that could reduce the by-effects, we tried to express and purify human cryptochrome 1 (hCRY1). The coding sequence of hCRY1 was inserted into prokaryotic expression plasmid pET28a(+), and this protein was purified from Escherichia coli BL21(DE3) after IPTG induction, ultrasonication, inclusion body dissolution, gradient dialysis, nickel column purification and ultrafiltration. The yield of hCRY1 in 1 L E. coli culture (LB medium) was about 10-15 mg. The radiation protective efficiency of hCRY1 was monitored by detecting X-ray-induced H2A.X foci in HaCaT cells. The results of immunofluorescence show that hCRY1 significantly reduces X-ray stimulated DNA damage response. The apoptosis of HaCaT cell was also detected, and the repression of H2A.X foci formation was not due to hCRY1's cytotoxity. All these data suggest a potential application of recombinant hCRY1 as a protective agent for radiotherapy.
Cryptochromes ; biosynthesis ; Escherichia coli ; Humans ; Plasmids ; Radiation-Protective Agents ; Recombinant Proteins ; biosynthesis

CFP10, ESAT6, Antigen 85A (Ag85A) and antigen 85B (Ag85B) are the key immunodominant antigens of Mycobacterium tuberculosis. In order to construct a eukaryotic vector able to co-express the four genes in one vector, we amplified the target gene fragments encoding the CFP10, ESAT6, Ag85A and Ag85B antigens and inserted them into the multicloning site of the shuttle plasmid vector pcDNA3.1 (+), of which the CFP10 and ESAT6 encoding genes were in frame fused with a linker encoding (Gly4Ser)3 residue, before the fused gene was inserted downstream of CMV promoter with a bovine growth hormone poly A(BGH pA) sequence at the 3'-end; Ag85A and Ag85B encoding genes were fused with a separation of internal ribosome entry site (IRES) sequence before the fused gene cassette was inserted downstream of RSV promoter with a BGH pA sequence at the 3'-end. The final plasmid containing all four genes was confirmed by sequence analysis and designated as pcDNA-CFP10-ESAT6-Ag85A-Ag85B (pcDNA-CEAB). In order to verify the ability of this construct to express target proteins, we then transfected the recombinant plasmid into Human embryonic kidney (HEK) 293T cells and harvested the cell lysates, and the cell lysates were then separated by SDS-PAGE and subjected to Western blot analysis 48 h after transfection. All four of the target proteins were detected in the cell lysates against the respective specific antibodies, suggesting that we have successfully constructed a eukaryotic vector co-expressing the four immunodominant antigens of Mycobacterium tuberculosis, which lay a foundation for the further study of the immunogenicity and protective activity of the four antigens.
Acyltransferases ; biosynthesis ; Antigens, Bacterial ; biosynthesis ; Bacterial Proteins ; biosynthesis ; Genetic Vectors ; HEK293 Cells ; Humans ; Immunodominant Epitopes ; Mycobacterium tuberculosis ; Plasmids

Tissue factor pathway inhibitor (TFPI) is one of the major physiological inhibitors of the human blood coagulation cascade and may have great potential in the prevention and therapy of diseases caused by thrombus formation. In this study, recombinant human tissue factor was generated in E. coli containing a recombinant vector constructed by inserting TFPI cDNA into pGEX-2T vector. The generated recombinant TFPI (rTFPI) could be simply purified with glutathione-agarose affinity method and maintained its biological function in terms of inhibition of tissue factor and factor Xa.
Escherichia coli ; genetics ; metabolism ; Humans ; Lipoproteins ; biosynthesis ; genetics ; pharmacology ; Peptide Fragments ; biosynthesis ; genetics ; pharmacology ; Plasmids ; Recombinant Proteins ; biosynthesis ; pharmacology ; Transfection

OBJECTIVETo construct a recombinant Lactobacillus acidophilus that expresses high levels of Helicobacter pylori (Hp) adhesin Hp0410.

METHODSThe gene fragment encoding Hp0410 was amplified by PCR from the DNA of H. pylori NCTC11639 strain and cloned into the shuttle plasmid pMG36e to construct pMG36e-Hp0410, which was transformed into Lactobacillus acidophilus by electroporation. The target protein was confirmed with SDS-PAGE and silver nitrate staining and analyzed by Western blotting. The stability of the recombinant plasmid was assessed by drawing the growth curve of the recombinant Lactobacillus acidophilus.

RESULTSA 750-bp fragment was inserted into the pMG36e plasmid and transformed into Lactobacillus lactis. The transformed bacterium expressed the target protein with a relative molecular mass of about 34 kD. Western blotting confirmed that the expressed proteins could be recognized by the serum of patients with Hp infection. The recombinant plasmid pMG36e-Hp0410 exhibited good stability in the presence or absence of erythromycin.

CONCLUSIONSThe recombinant Lactobacillus acidophilus with high constitutive expression of Hp0410 has been constructed successfully.

Adhesins, Bacterial ; biosynthesis ; genetics ; immunology ; Bacterial Vaccines ; biosynthesis ; Helicobacter Infections ; prevention & control ; Humans ; Lactobacillus acidophilus ; genetics ; metabolism ; Plasmids ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Vaccines, Attenuated ; biosynthesis

This study was conducted to amplify the cfpl0-esat6 fusion gene by SOE and insert into the integrating shuttle plasmid pMV361 to form the recombinant plasmid. Then another recombinant plasmid was constructed by insertinga-A g signal sequence of BCG. The two recombinant plasmids were introduced into BCG and the induced products from recombinant BCG were analyzed. In conclusion,the successful construction of rBCG expressing the fusion protein CFP10-ESAT6 will be the base of the development of novel Mycobacterium tuberclosis vaccines.
Antigens, Bacterial ; biosynthesis ; genetics ; Bacterial Proteins ; biosynthesis ; genetics ; Mycobacterium bovis ; genetics ; metabolism ; Mycobacterium tuberculosis ; genetics ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Tuberculosis Vaccines ; biosynthesis

OBJECTIVETo construct the recombinant vectors that express mouse Dishevelled 2 (Dvl2), and to evaluate its expression level in transfected RAW264.7 cells.

METHODSA pair of specific primers were designed according to the mouse Dvl2 cDNA sequence published in GenBank. Total RNA of RAW264.7 cells was extracted, and open reading frame of Dvl2 was obtained by RT-PCR, which was then cloned into pEZ-M29 plasmid. Electrophoresis after macrorestriction and DNA sequence analysis were used to identify the reconstructed plasmids. After transient transfection via liposome, the transfection of RAW264.7 cells was confirmed under a fluorescence microscope, and the expression level of Dvl2 was evaluated by real-time RT-PCR.

RESULTSThe recombinant plasmid containing mouse Dvl2, namely pEZ-M29/ Dvl2, was successfully constructed, and confirmed by DNA sequence analysis. After 48 h of tranfection, the expression of enhanced green fluorescene protein was observed under a fluorescence microscope, and real-time RT-PCR analysis revealed that the Dvl2 mRNA level was prominantly elevated in the transient transfected RAW264.7 cells.

CONCLUSIONThe recombinant plasmids pEZ-M29/Dvl2 are successfully constructed and can elevate the Dvl2 mRNA level in the transient transfected RAW264.7 cells, which can be used in further studies aiming at revealing the functional significance of Dvl2 in the osteoclastogenesis.

Adaptor Proteins, Signal Transducing ; biosynthesis ; Animals ; Cell Line, Tumor ; Dishevelled Proteins ; Genetic Vectors ; Mice ; Phosphoproteins ; biosynthesis ; Plasmids ; RNA, Messenger ; Transfection

BACKGROUNDTo clone the type common antigen gD of human herpes simplex virus I, II (HSV-I, II), the authors constructed recombinant expression vector Pmal-c2/gD and induced to express the fusion protein MBP-gD.

METHODSThe authors extracted HSV DNA,amplified gD gene by PCR assay and directly cloned it into prokaryotic expression vector pMAL-c2, then transformed it into E.coli DH5alpha. After proved to be correct by PCR, double enzyme digestion and sequencing, the fusion protein is induced to express by IPTG and detected by both Western blot and ELISA.

RESULTSThe constructed expression vector pMAL-c2/gD can be expressed with high efficiency. The product expressed was about 35.5% of the total bacterium proteins by SDS?PAGE analysis and was found nearly 39% as soluble protein,61% as inclusion in cytoplasm.

CONCLUSIONSThe authors constructed recombinant expression vector pMAL-c2/gD, the Western blotting result showed that the recombinant protein could be identified with gD specific monoclonal antibody DL6. Therefore the protein was of natural antigenic structure of gD.

Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Plasmids ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; Viral Envelope Proteins ; biosynthesis ; genetics

Based on site-specific transposition of an expression cassette into a baculovirus shuttle vector (Bacmid) which propagated in Escherichia coli, the Bac-to-Bac System provides a rapid and efficient method to generate recombinant baculoviruses and is widely used for high level expression of heterologous proteins. And the efficiency of recombinant baculovirus infecting cells plays an important role on the protein expression. In this study, we introduced an EGFP expression cassette driven by polyhedrin promoter into the p74 locus of Bacmid by homologous recombination. The target Bacmid-egfp was then transformed into E. coli DH10B containing the transposition helper plasmid to gain a new transposition receipt strain E. coli DH10Bac-egfp. Because of the intact attTn7 sites and lacZ', target gene cloned in a pFastBac vector can be transposed into the Bacmid-egfp shutter vector to construct recombinant baculovirus, which would allow the tracing of the target protein expression and the recombinant Bacmid transfection or recombinant baculoviral infection under fluorescence microscopes. Recombinant virus Bac-egfp-DsRed was constructed by transposing DsRed into the Bacmid-egfp in E. coliDHl0Bac-egfp, and the Sf9 cells infected with the recombinant virus expressed DsRed and EGFP efficiently. Another protein IL-6 fused with 6 x his tag was expressed and purified sucessfully from Sf9 cells infected with recombinant virus Bac-egfp-6 x his-IL6 constructed by the improved Bac-to-Bac system.
Animals ; Baculoviridae ; genetics ; Green Fluorescent Proteins ; genetics ; Interleukin-6 ; biosynthesis ; genetics ; Plasmids ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; Spodoptera