M2 protein of type A influenza virus is a good candidate for universal influenza vaccine, exotoxin A of Pseudomonas aeruginosa may facilitate the immunogenicity of M2 protein. We constructed and expressed a prokaryotic expression plasmid containing a chimeric gene of M2 extracellular coding region and a partial PEA gene, and observed the immunoprotection in BALB/c mice vaccinated with the fusion protein. The fusion protein (ntPE-M2e) was generated by inserting the coding sequence of the M2e in place of Ib loop in PEA. This fusion protein was used to immunize BALB/c mice by subcutaneously injection with incomplete Freund's adjuvant and boost at weeks 3 and 7. The immunized mice were challenged with influenza virus strain A/PR/34/8. The fusion protein (ntPE-M2e) immunization protected mice against lethal viral challenge. ELISA and ELISPOT results demonstrated that the fusion protein could induce a strong systemic immune response against synthetic M2e peptide, and virus replication in the lungs of mice was inhibited in comparison with the control. This study provides foundation for developing broad-spectrum vaccines against type A influenza viruses.
ADP Ribose Transferases ; genetics ; Animals ; Bacterial Toxins ; genetics ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Exotoxins ; genetics ; Female ; Gene Expression ; Immunization ; Influenza A virus ; immunology ; physiology ; Lung ; immunology ; virology ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; isolation & purification ; Viral Matrix Proteins ; biosynthesis ; genetics ; immunology ; isolation & purification ; Virulence Factors ; genetics

The M protein and streptococcus pyrogenic exotoxin (SPE A) are important virulence factors in group A streptococci (GAS) infections. The emm types of GAS strains isolated from patients with sepsis were determined by sequencing the 5' N-terminus of the emm gene, encoding the M protein, and clonality analysis using pulsed-field gel electrophoresis. The presence of speA and production of SPE A were also examined. There were no predominant GAS clones. The emm genotypes were variable, and the most common genotype was emm13 (17.9%). The production prevalence of SPE A was 21.4%. The low mortality rate (7.1%) of GAS sepsis might be attributable to the low incidence of virulent strains such as emm1 (10.7%) and emm3 (7.1%), as well as to low production rate of SPE A.
Virulence/genetics ; Variation (Genetics) ; Streptococcus pyogenes/*genetics/*pathogenicity ; Streptococcal Infections/*etiology/microbiology ; Sepsis/*etiology/microbiology ; Middle Aged ; Membrane Proteins/*biosynthesis/*genetics ; Male ; Infant ; Humans ; Genotype ; Female ; Exotoxins/*biosynthesis/*genetics ; DNA, Bacterial/genetics/isolation & purification ; Child, Preschool ; Child ; Bacterial Proteins/*biosynthesis/*genetics ; Aged ; Adult ; Adolescent

The objective of the experiment is to explore the purification and production of immunotoxin. The chimeric toxin, which is composed of 40 peptides of interleukin 10 (from amino acids 18 to 57) fused to a mutant form of Pseudomonas extoxin (PE) devoid of its native cell recognition domain. Two kinds of prokaryotic expression vector containing the chimeric toxin IL-10(18-57)-PE40 were constructed respectively. After induction of IPTG for 3 hours, IL-10(18-57)-PE40 was expressed highly in cytoplasmic fraction in Rosettablue(DE3), and was directed to periplasmic space as soluble form in E. coli BL21(DE3)pLysS . Western -blotting showed that the expressed protein could react with the specific rabbit sera against LHRH-PE40. With the application of salting out of (NH4)2SO4, hydrophobic interaction chromatography, Cu-affinity chromatography and anion exchange chromatography, the purity of IL-10(18-57)-PE40 was about 96%. The cytotoxicity assay, Cell-ELISA and fluorescent antibody test support the hypothesis that IL-10(18-57) based ligand-mediated cytotoxicity can serve to target cytotoxic agents in vitro.
ADP Ribose Transferases ; biosynthesis ; genetics ; Bacterial Toxins ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Exotoxins ; biosynthesis ; genetics ; Genetic Vectors ; Humans ; Immunotoxins ; genetics ; isolation & purification ; Interleukin-10 ; biosynthesis ; genetics ; Pseudomonas aeruginosa ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Virulence Factors ; biosynthesis ; genetics

IL-15 and IL-15 receptors (IL-15R) play a crucial role in the pathogenesis of adult T-cell leukemia (ATL), multiple myeloma and inflammatory autoimmune diseases. To develop a novel therapeutic agent capable of eliminating IL-15R-over-expressing abnormal cells, the gene coding for human IL-15 antagonist (IL-15M) was fused with a DNA fragment coding for the mutated form of Pseudomonas exotoxin, PEdelta293. The resulting gene fusion was cloned into pET16b under the control of T7 promoter, giving rise to the expression plasmid pET-IL15M-PEdelta293. Using Ni2+ -NTA affinity chromatography, IL15M-PEdelta293 was purified from E. coli BL21 (DE3) pLysS transformed with pET-IL15M-PEdelta293. The fusion toxin showed cytotoxicity to IL-15R-bearing myelogenous leukemia cell line K562 and K562-derived multidrug resistant cell line K562/AO2. However, IL-15R negative cell line Jurkat was insensitive to IL15M-PEdelta293. In addition, the toxic effect of IL15M-PEdelta293 on K562 was completely blocked by excessive amount of recombinant human IL-15. These results demonstrated that the selective cytotoxicity of IL15M-PEdelta293 correlated with the appropriate IL-15R expression on target cells. The present data suggest that the chimeric toxin constructed in this report may have therapeutic potential in the treatment of diseases associated with abnormal expression of IL-15/IL-15R, even in the treatment of chemotherapy refractory tumors.
Escherichia coli ; genetics ; metabolism ; Exotoxins ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; metabolism ; Humans ; Interleukin-15 ; antagonists & inhibitors ; biosynthesis ; genetics ; K562 Cells ; Pseudomonas aeruginosa ; genetics ; metabolism ; Receptors, Interleukin-15 ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

A recombinant immunotoxin named CEA/PE38/KDEL was constructed, which was composed of anti-CEA single-chain Fv and the truncated and modified form of Pseudomonas exotoxin (PE38/KDEL). The CEA/PE38/KDEL immunotoxin was expressed in the E. coli strain BL21 (DE3)-star as inclusion bodies. The denatured inclusion bodies were purified with Ni-NTA chelate agarose, then the constant gradient dialysis was used to perform the refolding of the CEA/PE38/KDEL immunotoxin. Results of FACS and MTT assay indicate that the refolded immunotoxins keep potent and specific cytotoxicity to tumor cells bearing CEA antigens.
ADP Ribose Transferases ; biosynthesis ; genetics ; pharmacology ; Antibodies ; genetics ; metabolism ; pharmacology ; Antineoplastic Agents ; metabolism ; pharmacology ; Bacterial Toxins ; biosynthesis ; genetics ; pharmacology ; Carcinoembryonic Antigen ; immunology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Exotoxins ; biosynthesis ; genetics ; pharmacology ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; Immunotoxins ; genetics ; isolation & purification ; metabolism ; pharmacology ; Protein Renaturation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Virulence Factors ; biosynthesis ; genetics ; pharmacology