Escherichia coli ; genetics ; Humans ; Recombinant Fusion Proteins ; immunology ; Viral Nonstructural Proteins ; genetics ; immunology

OBJECTIVETo express a multi-copy specific epitope recombinant protein of herpes simplex virus type 1 (HSV-1) with immuno-reactivity.

METHODSMulti-copy genes with a specific epitope of HSV-1-glycoprotein G 112-127 were constructed by DNA recombination and cloned in E. coli JM109 pGEM-5Zf. The positive recombinants were determined by SDS-PAGE and Western blotting.

RESULTSThe recombinants with 4, 8, 16 and 32 copies of gG 112-127 were obtained. The 8-copy recombinant was expressed by 17.5%, mainly as inclusion body. And it reacted with antiserum HSV-1, but not with antiserum HSV-2.

CONCLUSIONThe HSV-1-gG112-127 recombinant could be used to distinguish HSV-1 and HSV-2 in ELISA.

Antigen-Antibody Reactions ; DNA, Recombinant ; Epitopes ; biosynthesis ; immunology ; Herpesvirus 1, Human ; immunology ; Herpesvirus 2, Human ; immunology ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; immunology ; Viral Envelope Proteins ; biosynthesis ; immunology

Chimeric antigen receptors (CAR) are fusion proteins between single-chain variable fragments (scFv) from monoclonal antibodies and signaling domains of T-cells, which allow T-cells recognize specific cell-surface targets in an MHC-unrestricted fashion. The structure of CAR has changed over time, from first generation CAR (scFv + signaling moiety) to 2 and 3 generation CAR (combined with one or multiple costimulatory endodomains, such as CD28, 4-1BB and OX40), which enhance persistence, expansion and cytotoxicity of CAR. Many clinical trials treating hematological malignancies using the CAR-modified T-cells targeting CD19 and CD20 are under evaluation or even finished. These clinical trials indicated that CAR-based immunotherapy prolonged the survival of patients with relapsed/refractory B-cell malignancies. Furthermore, CAR have being studied to translate to other fields like adoptive therapy after hematopoietic stem cell transplantation. As to the treatment toxicity, CAR modified T-cell infusion is tolerant and safe in most patients. However, insertional mutagenesis, off-target effect and inflammatory response are safety issues surrounding CAR-modified T-cell therapy. In this review, the use of CAR technique in treatment of hematologic malignancies and evaluation of CAR safety are summarized.
Cell- and Tissue-Based Therapy ; Hematologic Neoplasms ; therapy ; Humans ; Immunotherapy ; Receptors, Antigen ; immunology ; Recombinant Fusion Proteins ; immunology ; T-Lymphocytes ; immunology

Cloning, Molecular ; Escherichia coli ; genetics ; Hepacivirus ; genetics ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Viral Nonstructural Proteins ; biosynthesis ; genetics ; immunology

Foot-and-mouth disease virus (FMDV) is the aetiological angent of a highly contagious viral disease. The complete gene encoding the structural protein of FMDV (P1) was subcloned into expression vector pGEX-KG, resulting in the fusion expression plasmid pKG-P1. After transformed into E. coli BL21(DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-P1 fusion protein was expressed in high level. The molecular weight of the fusion protein wa 110kD and the expressed products were soluble. Western-blotting was performed to confirm that the expressed fusion protein could specifically react with antiserum against FMDV. The fusion proteins were further purified by GST purification kit and an indirect ELISA (P1-ELISA) based on the purified proteins was developed. Comparison between P1-ELISA and the standard indirect haemagglutinin assay showed the two methods had 87 per cent agreement by detecting 864 serum samples, indicating the purified P1 protein was specific as the antigen of indirect P1-ELISA.
Capsid Proteins ; biosynthesis ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Foot-and-Mouth Disease Virus ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

OBJECTIVETo provide experimental evidence for development of human cytomegalovirus (HCMV) nucleic acid vaccine, HCMV surface protein (gB), membrane protein (pp150), and gB-pp150 fused gene eukaryotic expression vector were constructed.

METHODSgB and pp150 genes were amplified and fused into gB-pp150, then were cloned into pcDNA 3.1 (+) to obtain recombinant expression plasmids pcDNA 3.1 (+) -gB, pcDNA 3.1 (+) -pp150 and pcDNA 3.1 (+) -gB-pp150, which were encapsulated with chitosan. Mouse were vaccinated and the humoral and cell immune response were determined by ELISA, specific proliferative response of plenic lymphocytes.

RESULTSThe gB, pp150 and gB-pp150 fusion gene eukaryotic expression vector were successfully constructed. The antibodies A value induced by pcDNA3.1(+) -gB or pcDNA3.1 (+) -gB-pp150 were much higher than that of pcDNA3.1 (+) (P < 0.01). The IFN-gamma levels induced by pcDNA3.1 (+) -pp150 and pcDNA3.1 (+) -gB-pp150 were significantly higher than that of pcDNA3.1 (+). There are significant diference between the stimulating indexes of pcDNA3.1(+) -pp150 or pcDNA3.1 (+) -gB-pp150 immunized and normal mice.

CONCLUSIONThe DNA vaccine pcDNA3.1 (+) -gB can induce significant humoral immunity response, and pcDNA3.1 (+) -pp150 can induce high cellular immune response, whereas pcDNA3.1 (+) -gB-pp150 can induce both humoral and cellar immune responses in BALB/c mice.

Animals ; Cytomegalovirus ; genetics ; immunology ; Humans ; Immunity, Cellular ; immunology ; Immunization ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; immunology ; Vaccination ; Vaccines, DNA ; immunology ; Viral Envelope Proteins ; immunology

OBJECTIVEOf this study was to prepare high sensitivity and high specificity of highly pathogenic H5N1 subtype avian influenza virus NS1 protein antibody and a preliminary assessment of its potency.

METHODSConstruct pET-28a (+) recombinant vector containing the H5N1 subtype of avian influenza virus NS1 sequences of E. coli BL21 (DE3), induced expression of NS1 protein, NS1 recombinant protein was obtained by Ni-NTA column purified by affinity chromatography, and SDS-PAGE and Western Blot analysis. Purified protein antigen to immunize New Zealand white rabbits, obtained rabbit anti-NS1 serum, affinity-purified polyclonal antibodies. Using ELISA and Western Blot analysis of purified antibody titer and specificity.

RESULTSNS1 fusion protein was highly expressed in a purity of greater than 90%, with the fusion protein was used to immunize New Zealand white rabbits anti-NS1 polyclonal antibody titer of 1:80 000, and specific recognition of the H5N1 subtype of avian influenza virus NS1 protein.

CONCLUSIONSNS1 polyclonal antibodies to NS1 recombinant protein purified antigen, with better potency and specificity, and to prepare the conditions for the development of the H5N1 subtype of avian influenza virus detection kit.

Animals ; Antibodies, Viral ; biosynthesis ; immunology ; Escherichia coli ; genetics ; Influenza A Virus, H5N1 Subtype ; immunology ; Rabbits ; Recombinant Fusion Proteins ; immunology ; isolation & purification ; Viral Nonstructural Proteins ; genetics ; immunology

In the study, a gene encoding Tat protein N terminal 1- 21 amino acid residues-deleted mutant (Tat22-101) was amplified by PCR from a full length Tat gene of human immunodeficiency virus type 1, and the prokaryotic expression plasmid pET32a-Tat22-101 was constructed. After identification by digestion with endonucleases and sequencing, the recombinant plasmid pET32a-Tat22-101 was transformed into E. coli BL21(DE3) and expressed with IPTG induction. The mutant fusion protein with deleted Tat N terminal was purified by an affinity chromatography column Ni(2+)-NTA and subsequently identified by SDS-PAGE and Western blotting. The results showed that the molecular weight of the mutant protein was approximately 26.9kD. Furthermore, BALB/c mice were immunized with the mutant protein and the anti-sera were collected. ELISA results showed that the mutant protein preserved its immunogenicity, particularly it could improve the production of antibodies to other epitopes in addition to the N terminal epitope of Tat protein, which might provide some valuable information for the study of Tat functions as well as for development of potential novel HIV Tat vaccine.
Animals ; Female ; Gene Products, tat ; biosynthesis ; genetics ; immunology ; HIV-1 ; genetics ; immunology ; Humans ; Mice ; Mice, Inbred BALB C ; Mutant Proteins ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

OBJECTIVE: To synthesize the specific CagA gene segment of the gastric cancer idiotype Helicobacter pylori (H. pylori), establish the prokaryotic expression system and identify the antigenicity sequence of recombination signals. METHODS: We selected the CagA fragment which was related to gastric cancer in our earlier research. The CagA gene segment was optimized and synthesized. The synthesized CagA gene was cut from the pUC57-CagA plasmid and then was carried by expression vector pET32a to be transformed into the host bacterium BL21 (DE3). The positively cloned pET32a-CagA was selected by receptivity of aniline and colony PCR. The host bacterium with pET32a-CagA was induced by IPTG to express fusion protein. The expression of CagA protein was analyzed by SDS-PAGE gel electrophoresis and the antigenicity of fusion protein was examined by Western blot. RESULTS: CagA gene segment was designed and synthesized. The sequence of synthesis CagA gene segment was the same as the one designed before (AF289435). We successfully constructed the plasmid of prokaryotic expression of the pET32a-CagA. Homology of the target CagA proteinum was 100%, the same as AAG09884. The host bacterium BL21 (DE3) containing pET32a-CagA could express CagA fusion protein after the IPTG induction. SDS-PAGE gel electrophoresis showed that the molecular weight of fusion protein was the same as expected (45 kD). Western blot showed that the fusion protein could be combined with the antibody of the whole bacterium of anti-H. pylori. CONCLUSION The synthesized CagA fusion protein from the prokaryotic expression system has antigenicity. We hope to set the foundation for selecting the strain in H. pylori correlated to gastric cancer and corresponding therapy in clinical practice.
Amino Acid Sequence ; Antigens, Bacterial ; genetics ; immunology ; Bacterial Proteins ; genetics ; immunology ; Base Sequence ; Genetic Vectors ; Helicobacter pylori ; genetics ; immunology ; Molecular Sequence Data ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

Adenoviridae ; genetics ; Animals ; Dengue Vaccines ; immunology ; Humans ; Plasmids ; Recombinant Fusion Proteins ; immunology ; Vaccines, Attenuated ; immunology ; Vaccines, DNA ; immunology ; Vaccines, Synthetic ; immunology