To enhance the efficiency of the expression of target gene in eukaryotic cells, one of the strongest prokaryotic expression systems, the T7 RNA polymerase and T7 promoter, was introduced into eukaryotic cells. A duel-plasmid gene expression system of T7 bacteriophage components was developed; one containing the T7 phage RNA polymerase gene under the control of eukaryotic promoter CMV (pCMV-T7pol) and the other (pT7IRES) containing the T7 promoter and T7 terminator as well as EMCV IRES. To test the feasibility of this plasmid system for eukaryotic expression, hepatitis B virus envelop HBV preS2/S was used to construct pT7IRES-HBs. The target genes were expressed efficiently by the eukaryonized prokaryotic expression system in a variety of the cells indicating C2C12, SP2/0, NIH3T3 and BALB/c 3T3, suggesting the potential applications of the expression system in gene therapy and gene immunization.
Animals ; Bacteriophage T7 ; genetics ; Cell Line ; DNA-Directed RNA Polymerases ; genetics ; Gene Targeting ; Promoter Regions, Genetic ; genetics ; Viral Proteins ; genetics

Prospect Hill virus (PHV) is related antigenically but distinct to Hantaan virus. As a member of genus Hantavirus, PHV has three segmented RNA genome. Among these segments, Small segment(S) encodes nucleocapsid protein (NP) as structural protein and also may do functional nonstructural protein(NSs). We performed in vitro transcription to produce vRNA of PHV S genome. For the first step of in vitro transcription of S genome of PHV, the S RNA segrnent which is 1,675 nucleotides long was amplified by RT-PCR using PCR primers built according to cDNA sequence of PHV S genome. Next, a new PCR primer appended above downstream primer to T7 phage promoter sequence was reconstructed to obtain PCR product containing T7 promoter. Then another PCR was performed. Using this PCR product as the template, in vitro run-off transcription without cloning by transcriptional vector was performed to obtain viral- sense RNA transcript. Thereafter, the size of transcript was assessed by running on formaldehyde agarose gels. Since the transcription reactants contain a-S UTP, the transcript is detectable by autoradiography. The transcript was also detectable by northern hybridization with a-P dCTP- labelled PHV amplicon probe (319 bp) and the initiation start point of run-off transcription was also determined by primer extention analysis. Our data indicate that the in vitro transcript could be produced from the PCR product amplified by PCR primer containing T7 phage promoter without cloning into a phage transcription vector.
Autoradiography ; Bacteriophage T7 ; Bacteriophages ; Clone Cells ; Cloning, Organism ; DNA, Complementary ; Formaldehyde ; Gels ; Genome* ; Hantaan virus ; Hantavirus ; Nucleocapsid Proteins ; Nucleotides ; Polymerase Chain Reaction ; RNA ; Running ; Sepharose ; Uridine Triphosphate ; Viruses*

The gene encoding E7 oncoprotein of human papillomavirus type 16 was cloned and expressed in Escherichia coli, and the monoclonal antibodies (Mabs) against this expressed protein were generated. For the efficient immunization, two kind of recombinant E7 protein in fusion form were produced. One was maltose binding protein (MBP) fusion type (MBP-E7) and the other was T7 phage gene 10 product fusioa type (gene 10-E7). Immunization with these two fusion protein to mice, finally two Mabs (VD6 and IB10) were obtained. VD6 and IB10 showed reactivities with E7 protein in CaSki cell but not in HeLa by Western blot analysis. In addition, the Mab, VD6, reacted with COS-7 cell transfected with E7 gene majorly in cytoplasm by immunofluorescence test. Also VD6 could detect E7 protein in cytoplasm and nucleus of CaSki ceU by immunogold electron microscopy. Based on these results, the Mab VD6 was could be used for various E7 detection system such as Western blot analysis and immunohistochemical methods.
Animals ; Antibodies, Monoclonal* ; Bacteriophage T7 ; Blotting, Western ; Clone Cells ; COS Cells ; Cytoplasm ; Escherichia coli ; Fluorescent Antibody Technique ; Humans* ; Immunization ; Maltose-Binding Proteins ; Mice ; Microscopy, Electron

To make transcription of the target gene be driven by T7 RNA polymerase (T7 RNAP) in the eukaryotic cells, and the transcripts be CAP-independent translated. Firstly, the T7 RNAP was introduced into eukaryotic cells by two methods: (1) the BHK-21 cells were contransfected by the plasmid expressing T7 RNAP and pIERS-EGFP-ET vector; (2) by transfection of the cell line stably expressing T7 RNAP. The internal ribosome entry site (IRES) element from FMDV was cloned into the downstream of the T7 promoter sequence of the prokaryotic expressing vector pET-40a-c (+), resulted in the plasmid would express the transcripts carrying the IERS element at its 5' end. The enhanced green fluorescent protein (EGFP) gene was cloned into the downstream of the IERS element, resulted in plasmid pIERS-EGFP-ET. Then, the two kinds of cells expressing T7 RANP were transfected by pIERS-EGFP-ET. The green fluorescence in the transfected cells was observed under a fluorescence microscope equipped with a video documentation system. And the expressional efficiency was analyzed with flow cytometry (FCM). The results show that the IRES element from FMDV has the role of initiating CAP-independent translation, and lay foundation for researching function of the element and interrelated proteins. It would be potential for expressing target gene by the T7 RNAP couple expression system.
Bacteriophage T7 ; enzymology ; genetics ; Cloning, Molecular ; DNA-Directed RNA Polymerases ; biosynthesis ; genetics ; Foot-and-Mouth Disease Virus ; genetics ; Genes, Viral ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Transfection ; Viral Proteins ; biosynthesis ; genetics

OBJECTIVETo increase the immune effect of gene vaccine, T7 RNA polymerase was used to establish a system of cytoplasmic expression.

METHODS(1) The plasmid pT7 EMCVP1, including T7 promoter sequence, 5'-untranslated sequence of encephalomyocarditis (EMC) virus, VP1 sequence of coxsackievirus B3 (CVB3), was cotransfected with the plasmid pAR 3132, which codes for the T7 RNA polymerase, into HeLa cells and murine peritoneal macrophages. (2) The plasmid pT7 EMCVP1 and pAR 3132 were respectively transformed into the attenuated Salmonella typhimurium SL 7207. The two kinds of transformed bacteria were coinfected into murine peritoneal macrophages.

RESULTS(1) The target antigen VP1 in the cytoplasm was about 2-4-fold higher than that of pcDNA3 VP1 singly transfected. (2) After the murine peritoneal macrophages were coinfected by two kinds of transformed bacteria, the target antigen VP1 could also be detected.

CONCLUSIONThe pT7 EMCVP1 and pAR 3132 could be expressed in the cytoplasm of HeLa cells and murine peritoneal macrophages and the amount of the antigen VP1 increased remarkably as compared with that of pcDNA3 VP1 singly transfected.

Animals ; Bacteriophage T7 ; genetics ; Capsid Proteins ; genetics ; metabolism ; Cytoplasm ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Enterovirus B, Human ; genetics ; Gene Expression ; HeLa Cells ; Humans ; Macrophages, Peritoneal ; cytology ; metabolism ; Mice ; Plasmids ; genetics ; Promoter Regions, Genetic ; genetics ; Salmonella typhimurium ; genetics ; Transfection

Microtus fortis is naturally resisitent to Schistosoma japonicum. In order to find schistosome-resistence-related genes of Microtus fortis, a T7 phage-display cDNA library from liver of Microtus fortis was screened with the soluble lysate of schistosomula. The specific phages were enriched 375-fold after 3 rounds of biopanning. Ninety-two positive clones picked at random were sequenced and 19 ESTs including 6 unreported genes were obtained. Compared with the negative phage clone control, five positive clones, No.4 (GenBank Accession No.: EW968294), No.13 (GenBank Accession No.: EW968303), No.14 (GenBank Accession No.: EW968304), No.15 (GenBank Accession No.: EW968305) and No.18 (GenBank Accession No.: EW968308) could induce significantly higher schistosomula mortality rate when co-cultivated with schistosomula. According to the function analysis and the shistosomula-killing effect in vitro, the genes encoding CASP8 and FADD-like apoptosis regulator isoform protein, alpha-2-HS-glycoprotein, M4 protein, R3H domain (binds single-stranded nucleic acids) isoform 2 and 3 previously unreported proteins (No.14, No.15 and No.18) obtained here, were schistosomiasis-resistence-related genes of Microtus fortis.
Animals ; Arvicolinae ; genetics ; parasitology ; Bacteriophage T7 ; genetics ; Cloning, Molecular ; Expressed Sequence Tags ; Gene Library ; Genes, Helminth ; genetics ; Immunity, Innate ; genetics ; Larva ; genetics ; growth & development ; Liver ; chemistry ; Schistosoma japonicum ; genetics ; growth & development

In order to realize over-expression of Burkholderia cepacia (B. cepacia) lipase, we introduced the widely used T7 RAN polymerase expression system into B. cepacia G63 to over-express the lipase gene. By using PCR technique, we amplified the T7 RNA polymerase gene (T7 RNAP) from the BL21 (DE3) and cloned it into the suicide plasmid pJQ200SK. After that, we flanked T7 RNAP with two 500 bp homologous fragments and integrated it into the genomes of B. cepacia by tri-parental mating, so that T7 RNAP was under-controlled by lipase gene (lipA) promoter. Then, we cloned the lipA and its partner gene lipB into the vector pUCPCM and pBBR22b both or separately. Therefore, we got 7 expression plasmids pBBR22blipAB, pBBR22blipA, pUCPCMlipAB, pUCPCMlipA, pUCPCMdeltalipAlipB, pUCPCMdeltalipA, pUCPCMdeltalipB, and then electroporated them into B. cepacia containing T7 RNA. After shake flask culture, we found B. cepacia containing pUCPCMlipAB produced the most quantity of lipase, and lipase activity was up to 607.2 U/mg, 2.8-folds higher than that of the wild strain. Moreover, lipase activities of all engineering strains except the one containing pUCPCMdeltalipB were enhanced to some extent. The specific activities of wild type B. cepacia and B. cepacia containing pUCPCMlipAB were respectively 29 984 U/mg and 30 875 U/mg after ammonium sulfate precipitation and gel filtration chromatography. The T7 RNA polymerase expression system could effectively enhanced lipase expression in B. cepacia, and secretion signal PelB and ribosome-binding site may promote lipase expression in engineering strain.
Bacteriophage T7 ; genetics ; Burkholderia cepacia ; enzymology ; genetics ; Cloning, Molecular ; DNA-Directed RNA Polymerases ; genetics ; Escherichia coli ; enzymology ; genetics ; Lipase ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transformation, Genetic ; Viral Proteins ; genetics

To construct a recombinant T7 phage expressing matrix protein 2 ectodomain (M2e) peptides of avian influenza A virus and test immunological and protective efficacy in the immunized SPF chickens. M2e gene sequence was obtained from Genbank and two copies of M2e gene were artificially synthesised, the M2e gene was then cloned into the T7 select 415-1b phage in the multiple cloning sites to construct the recombinant phage T7-M2e. The positive recombinant phage was identified by PCR and sequencing, and the expression of surface fusion protein was confirmed by SDS-PAGE and Western-blot. SPF chickens were subcutaneously injected with 1 X 10(10) pfu phage T7-M2e, sera samples were collected pre- and post-vaccination, and were tested for anti-M2e antibody by ELISA. The binding capacity of serum to virus was also examined by indirect immunofluorescence assay in virus- infected CEF. The immunized chickens were challenged with 200 EID50 of H9 type avian influenza virus and viral isolation rate was calculated to evaluate the immune protective efficacy. A recombinant T7 phage was obtained displaying M2e peptides of avian influenza A virus, and the fusion protein had favorable immunoreactivity. All chickens developed a certain amount of anti-M2e antibody which could specially bind to the viral particles. In addition, the protection efficacy of phage T7-M2e vaccine against H9 type avian influenza viruses was 4/5 (80%). These results indicate that the recombinant T7 phage displaying M2e peptides of avian influenza A virus has a great potential to be developed into a novel vaccine for the prevention of avian influenza infection.
Animals ; Antibodies, Viral ; blood ; Bacteriophage T7 ; genetics ; immunology ; metabolism ; Chickens ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Regulation, Viral ; Immunization ; Influenza A virus ; genetics ; immunology ; Influenza Vaccines ; immunology ; Influenza in Birds ; immunology ; metabolism ; prevention & control ; Peptides ; genetics ; immunology ; metabolism ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; Specific Pathogen-Free Organisms ; Viral Matrix Proteins ; genetics ; immunology ; metabolism

OBJECTIVETo construct a recombined phage vaccine and to evaluate the efficiency of this phage vaccine against EGFR-positive tumors.

METHODST7 phage display system was used to display five fragments of the extracellular domain of chicken EGFR. The EGFR was expressed as a fused protein on the surface of the T7 phage 10B capsid protein. The EGFR expression of the phage vaccine was verified by Western-blot analysis. Anti-EGFR antibody was detected by ELISA. Splenic lymphocytes of the immunized mice were separated and used to determine the immunotoxic effect against A431 cells. The phage vaccines were injected into C57 mice 4 times before Lewis lung cancer cells inoculation. Tumor volume was recorded to evaluate the anti-tumor effect of each vaccine.

RESULTSFive phage vaccines inserted with the chicken EGFR gene were successfully constructed. Western blot assay showed that the extracellular domain of chicken EGFR proteins were displayed on the surface of the phage. Specific antibody was induced in the immunized mice, compared with the control group. Splenic lymphocytes of the immunized mice were shown to be immunotoxic against A431 cells. The killing rates of the experimental groups were higher than that of control group (P < 0.001, t-Student test). The highest killing rate was (45.74 +/- 7.21)%. The tumor growth was inhibited in the experimental groups compared with those of control groups (P < 0.05 in C1, C2, C3, C4 groups, P > 0.05 in C5 group).

CONCLUSIONOur results demonstrated that recombined EGFR phage vaccines may be used to induce therapeutic anti-tumor immunity against EGFR-positive tumors.

Animals ; Bacteriophage T7 ; genetics ; Blotting, Western ; Cancer Vaccines ; administration & dosage ; genetics ; immunology ; Capsid Proteins ; genetics ; metabolism ; Carcinoma, Lewis Lung ; immunology ; pathology ; therapy ; Cell Line, Tumor ; Chickens ; Cytotoxicity Tests, Immunologic ; Immunotherapy ; methods ; Male ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Random Allocation ; Receptor, Epidermal Growth Factor ; genetics ; immunology ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism