Constructing ethanologenic strains with cellulose activity is important to achieve consolidated bioprocessing of lignocellulose for ethanol production. In this study, we integrated the pyruvate decarboxylase gene pdc and alcohol dehydrogenase gene adhB from Zymomonas mobilis ZM4 into Escherichia coli JM109 by Red recombination method to generatea recombinant strain E. coli P81 that could produce ethanol from glucose. Abeta-glucosidase gene bglB from Bacillus polymyxa 1.794 was cloned into the recombinant E. coli P81 and beta-glucosidase was expressed to give a new recombinant strain E. coli P81 (pUC19-bglB) with dual functions of cellobiose degradation and ethanol production. The extracellular beta-glucosidaseactivity was 84.78 mU/mL broth and the extracellular cellobiase activity of E. coli P81 (pUC19-bglB) was 32.32 mU/mL broth. E. coli P81 (pUC19-bglB) fermented cellobiose to ethanol with a yield of 55.8% of the theoretical value, and when glucose and cellobiose were co-fermented, the ethanol yield reached 46.5% of thetheoretical value. The construction of consolidated bioprocessing strain opens the possibility to convert cellobiose to ethanol in a single bioprocess.
Bacterial Secretion Systems ; Cellulose ; metabolism ; Escherichia coli ; genetics ; metabolism ; Ethanol ; metabolism ; Fermentation ; Recombinant Proteins ; biosynthesis ; genetics ; beta-Glucosidase ; biosynthesis ; genetics

To develop a method based on immunoreactions for detection of Ectropis obliqua Nucleopolyhedrovirus (EoNPV), the polyhedra of the virus were purified and used to immunize the mouse BALB/c. The spleen cells from the immunized mice were then fused with the myeloma cell line Sp2/0. A hybridoma cell line which can stably secrete the monoclonal antibody against EoNPV was achieved by using indirect ELISA screening and cloning methods, and was named as 7D3. Meanwhile, the polyhedrin gene was cloned from EoNPV and expressed in E. coli. Western blotting analysis showed that the monoclonal antibody prepared from 7D3 could specifically react with the recombinant polyhedrin. An indirect ELISA method based on this monoclonal antibody for detecting EoNPV in infected tea looper was developed.
Animals ; Antibodies, Monoclonal ; biosynthesis ; genetics ; immunology ; Antibody Specificity ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Hybridomas ; secretion ; Lepidoptera ; growth & development ; virology ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Viral Structural Proteins ; biosynthesis ; genetics ; immunology

Relative deficiency in production of glycoprotein hormone erythropoietin (Epo) is a major cause of renal anemia. This study planned to investigate whether the hypoxia-regulated system of Epo expression, constructed by fusing Epo gene to the chimeric phosphoglycerate kinase (PGK) hypoxia response elements (HRE) in combination with cytomegalovirus immediate-early (CMV IE) basal gene promoter and delivered by plasmid intramuscular injection, might provide a long-term physiologically regulated Epo secretion expression to correct the anemia in adenine-induced uremic rats. Plasmid vectors (pHRE-Epo) were synthesized by fusing human Epo cDNA to the HRE/CMV promoter. Hypoxia-inducible activity of this promoter was evaluated first in vitro and then in vivo in healthy and uremic rats (n = 30 per group). The vectors (pCMV-Epo) in which Epo expression was directed by a constitutive CMV gene promoter served as control. ANOVA and Student's t-test were used to analyze between-group differences. A high-level expression of Epo was induced by hypoxia in vitro and in vivo. Though both pHRE-Epo and pCMV-Epo corrected anemia, the hematocrit of the pCMV-Epo-treated rats exceeded the normal (P < 0.05), but that of the pHRE-Epo-treated rats didn't. Hypoxia-regulated system of Epo gene expression constructed by fusing Epo to the HRE/CMV promoter and delivered by plasmid intramuscular injection may provide a long-term and stable Epo expression and secretion in vivo to correct the anemia in adenine-induced uremic rats.
Anemia/blood/*therapy ; Animals ; Base Sequence ; Blood Urea Nitrogen ; Cell Hypoxia ; Creatinine/blood ; Erythropoietin/biosynthesis/*genetics/secretion ; Gene Expression Regulation ; Genes, Reporter ; Genetic Therapy ; HeLa Cells ; Humans ; Injections, Intramuscular ; Kidney/pathology ; Luciferases, Firefly/biosynthesis/genetics ; Molecular Sequence Data ; Plasmids/*genetics ; Promoter Regions, Genetic ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins/biosynthesis/genetics/secretion ; Response Elements ; Transcriptional Activation ; Uremia/blood/*therapy

Aspergillus niger is an important industrial workhorse with extensive application in the sectors of industrial enzymes, heterogeneous proteins, organic acids and etc. The disclosure of its genomic sequence to the public brought the study of A. niger into the post-genomic era. Diverse omic data are being produced massively and rapidly, which largely upgrades our understanding to the hyperproduction mechanism of A. niger to a systems and molecular level. At meanwhile, its genetic operating system is becoming mature, which enables genome-scale genetic perturbation within A. niger. In conclusion, we are on the right way to redesign and engineer A. niger to an omnipotent cellular factory.
Aspergillus niger ; genetics ; metabolism ; Biotechnology ; methods ; Enzymes ; genetics ; secretion ; Gene Expression Regulation, Fungal ; Genes, Fungal ; Genome, Fungal ; Protein Biosynthesis ; genetics ; Recombinant Proteins ; secretion ; Transcription, Genetic

OBJECTIVETo obtain the monoclonal antibody against hexon protein of human adenovirus.

METHODSBALB/c mice were immunized with purified recombinant hexon protein, and the spleen cells of the mice were isolated and fused with myloma cells. Four hybridoma cell strains were screened by indirect ELISA and cultured, and the sensitivity, specificity and virus neutralizing activity were analyzed with ELISA, Western blotting and neutralizing test.

RESULTSThe mouse ascites produced by these hybridoma cells contained specific monoclonal antibodies against hexon protein of human adenovirus as identified by ELISA and Western blot, and the antibody generated by 4C6 strain showed human adenovirus type 3-neutralizing activity.

CONCLUSIONThe monoclonal antibodies against hexon protein with high specificity have been successfully obtained, and these antibodies can be useful in developing assays for early diagnosis of HAdV3 infection and also in study of therapeutic drugs of the infection.

Adenoviruses, Human ; chemistry ; immunology ; Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibodies, Viral ; biosynthesis ; immunology ; Blotting, Western ; Capsid Proteins ; biosynthesis ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Humans ; Hybridomas ; secretion ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; immunology

The cDNA of Insulin-like growth factor binding protein 3 was subcloned into a eukaryotic secretory expression vector pSectagA to construct pSectag-IGFBP3. Human renal cell carcinoma (RCC) 786-0 cells were transfected with pSectag-IGFBP3 using lipofectamine 2000. After 48 h, the secretory IGFBP-3 was tested and identified by western blotting. Meanwhile, Annexin V-EGFP stain was used to analyze the apoptosis of 786-0 cells induced by IGFBP-3. Secretory IGFBP-3 protein could express successfully in the 786-0 cells and the expressed IGFBP-3 directly displayed an apoptotic effect on the host cells. This work provides a basis for further study on the apoptosis-inducing mechanism of IGFBP-3 and the development of a new anti-tumor drug.
Apoptosis ; drug effects ; Base Sequence ; Carcinoma, Renal Cell ; metabolism ; pathology ; Humans ; Insulin-Like Growth Factor Binding Protein 3 ; genetics ; secretion ; Kidney Neoplasms ; metabolism ; pathology ; Molecular Sequence Data ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; physiology ; Transfection ; Tumor Cells, Cultured

Based on the genomic sequence of SARS-CoV strain BJ101, antigenic immunodominant genes coding for the structure proteins of SARS-CoV were predicted by bio-informatics methods, and two chimeric genes A and B with multi-immunodominants lined up by Gly-Pro-Gly linker were synthesized. The chimeric genes were cloned into plasmid pGEX-6p-1 and expressed in E. coli with IPGT inducing. BALB/c mice were immunized with the purified recombinant fusion protein. The specificity of monoclonal antibodies were tested with a commercial ELISA kit for detecting antibody against SARS-CoV. The results showed that two peptides with molecular weights of 34kD and 35kD expressed by the two chimeric genes could be recognized by SARS patient convalescent serum in Western blot. Six positive hybridoma cell lines stably secreting monoclonal antibodies were selected. The subtype of monoclonal antibody D3C5 is IgG2a, and subtypes of all other five monoclonal antibodies are IgG1. Light chains of all monoclonal antibodies are kappa. With a commercial SARS-CoV antibodies detection ELISA kit, five out of six monoclonal antibodies were positively recognized. In western blot analysis with inactived virus cultures, D3D1 specifically recognized a band of about 180 kD. To further analyse the epitopes corresponding to the monoclonal antibodies, six oligoes (S1-S6) from S gene were synthesized and expressed. The results showed that the monoclonal antibodies D3D1 and D3C5 specifically recognized expression product of S2 and S5 oligoes, respectively. The S2 and S5 oligoes are corresponding to 447-458aa and 789-799aa of SARS-CoV S protein respectively.
Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibodies, Viral ; biosynthesis ; immunology ; Antibody Specificity ; Epitopes ; genetics ; Escherichia coli ; genetics ; metabolism ; Female ; Humans ; Hybridomas ; secretion ; Membrane Glycoproteins ; immunology ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; SARS Virus ; immunology ; Spike Glycoprotein, Coronavirus ; Viral Envelope Proteins ; immunology

OBJECTIVETo better understand the molecular mechanism of tumorigenesis and progression, the monoclonal antibody against G3BP (Ras-GAP SH3 binding protein), which serves as an important downstream effector of Ras signaling, was prepared, characterized and utilized in analysis of various human tumors.

METHODSBy using the prokaryotic expression vector pGEX-5X1, GST-G3BP fusion protein was expressed in E. coli BL21 under induction of IPTG. Purified GST-G3BP fusion protein was used to immunize BALB/c mice. The monoclonal antibody against G3BP was produced through conventional hybridoma method and characterized by ELISA, Western blot and immunohistochemical staining.

RESULTSA hybridoma cell line secreting anti-G3BP IgG1 subtype antibody was obtained. Western blot and competitive inhibition assay showed that the antibody was G3BP-specific. Immunohistochemical staining demonstrated that G3BP was over-expressed in formalin-fixed and paraffin-embedded tissues of some human tumors, such as lung cancer, colon cancer, gastric cancer and breast cancer. In breast cancer specimens, the degree of G3BP expression correlated positively with the presence of lymph node metastasis and c-erbB2 expression.

CONCLUSIONSThe G3BP-specific monoclonal antibody derived from recombination protein can be used in ELISA, Western blot and immunohistochemical assay. It may provide an important tool in analysis of G3BP in in vitro and in vivo experiments. Besides, G3BP may serve as another prognostic marker for breast cancer.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Biomarkers, Tumor ; Breast Neoplasms ; metabolism ; pathology ; Carrier Proteins ; genetics ; immunology ; metabolism ; DNA Helicases ; Female ; Genetic Vectors ; Humans ; Hybridomas ; secretion ; Lymphatic Metastasis ; Male ; Mice ; Mice, Inbred BALB C ; Poly-ADP-Ribose Binding Proteins ; RNA Helicases ; RNA Recognition Motif Proteins ; Receptor, ErbB-2 ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Tumor Cells, Cultured

OBJECTIVETo establish a cell model of secreted alkaline phosphatase (SEAP) co-controlled by HCV 5'NCR and NS3 serine protease in an effort to develop new antiviral agents.

METHODSThe fragments of HCV 5'NCR and NS3/4A-SEAP were amplified by PCR. They were fused into pBluescript SK+ to generate 5'NCR-NS3/4A-SEAP chimeric plasmid. The resulting chimeric gene was subcloned into HindIII/Bsu36 I site of pSEAP2-Control (a SEAP eukaryotic expression plasmid), to generate pNCR-NS3/4A-SEAP, in which the SEAP was fused in-frame to the downstream of NS4A/4B cleavage site. The SEAP activity in the culture media of transiently transfected cells was monitored quantitatively. The regulatory effect of HCV 5'NCR and NS3 serine protease on SEAP expression was measured by treatment of transfected cells with antisense oligodeoxynucleotide (ASODN) against HCV 5'NCR and TPCK, a irreversible serine protease inhibitor.

RESULTSThe SEAP activity in the culture media reached 80801+/-4794 RLU, and was significantly inhibited by 5 micromol/L, 10 micromol/L of ASODN (t=4.315, p<0.01; t=6.985, p<0.001) and 100 micromol/L of TPCK (t=6.949, P<0.001).

CONCLUSIONA cell model of SEAP co-controlled by HCV 5'NCR and NS3 serine protease has been successfully established. This might promote the screening of anti-viral drugs

Alkaline Phosphatase ; secretion ; Antiviral Agents ; Drug Evaluation, Preclinical ; Hepacivirus ; genetics ; Hepatocytes ; enzymology ; virology ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; Serine Endopeptidases ; biosynthesis ; genetics ; Viral Nonstructural Proteins ; biosynthesis ; genetics

OBJECTIVETo construct a DNA vaccine as a prophylactic model to prevent condyloma acuminatum and detect its immunogenicity in mice.

METHODSThe major capsid protein (L1) gene of human papillomavirus (HPV) 6b was inserted into an eukaryotic expression plasmid (pcDNA3.1). The recombinant plasmid was transfected into COS-7 cells. Western blot were performed to detect whether L1 protein can be expressed in eukaryotic cells. Eighteen female BALB/c mice were tested for immunogenicity study.

RESULTSThe recombinant plasmid (pcDNA3.1-HPV6bL1) was verified as HPV6b L1 gene by sequencing. Western blot showed specific strip. Anti-L1 protein antibodies could be detected in the mice's sera inoculated with pcDNA3.1-HPV6bL1. Similarly, IL-4, IL-2, and IFN-gamma were increased in the same mice.

CONCLUSIONHPV6b L1 recombinant plasmid was constructed successfully which had immunogenicity for BALB/c mice. It provided experimental evidence for the research of DNA vaccine of condyloma acuminata.

Animals ; Antibodies, Viral ; blood ; COS Cells ; Capsid Proteins ; Cercopithecus aethiops ; Condylomata Acuminata ; immunology ; prevention & control ; Female ; Humans ; Immunization ; Interferon-gamma ; secretion ; Interleukin-2 ; secretion ; Interleukin-4 ; secretion ; Mice ; Mice, Inbred BALB C ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; immunology ; Papillomaviridae ; genetics ; Plasmids ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection ; Vaccines, DNA ; immunology