Biological properties the novel application of N-trimethyl chitosan nanospheres as a stabilizer and preservative in tetanus vaccine

Purpose@#Chitosan is a natural polymer that has excellent properties include biocompatibility, biodegradability, no cytotoxicity, high charge density, low cost, mucoadhesive, permeation enhancing (ability to cross tight junction), and immunomodulating ability that makes the spectrum of its applicability much broader. This study was conducted to investigate the stabilizing, preservative and immunogenicity properties of N-trimethyl chitosan nanospheres (N-TMCNS). @*Materials and Methods@#The tetanus toxoid (TT) was encapsulated into N-TMCNS and then characterized by scanning electron microscope, atomic force microscope, and dynamic light scattering. For stabilizer assay of N-TMCNS after storage of TT-N-TMCNS at different temperatures for 3 weeks, they were used for immunization of mice and different temperatures groups’ anti-TT-N-TMCNS production compared with other groups. Finally, the immunized mice were challenged with tetanus toxin. The preservation activity of TT-N-TMCNS against Escherichia coli was compared with thimerosal formulated TT. @*Results@#Our results revealed that heat-treated TT-N-TMCNS could induce higher titer of neutralizing immunoglobulin G in compared to TT vaccine and was able to protect the mice better than TT vaccine in challenge test. Furthermore, N-TMCNS as a preservative inhibited the growth of E. coli more effective than thimerosal. @*Conclusion@#Overall, the obtained results indicated that the N-TMCNS is one of the best stabilizer and preservative agent that can be used in the formulation of TT vaccine.

[Production of antibody against CtxB recombinant protein based on nanofibers and nanocapsules]

Objectives: CtxB [Cholera toxin B subunit] contributes to a vaccine's efficacy by stimulating production of the anti-CtxB antibody. Various attempts have been made to increase production of this antibody. Chitosan is a mucoadhesive polysaccharide that has tremendous potential for oral vaccine delivery in terms of its exclusive features that include biocompatibility, biodegradability, high charge density and non-toxicity. We investigated the potential for chitosan nanofibers and nanocapsules as novel carrier systems for the oral delivery of CtxB

Methods: Antigen-containing chitosan nanofibers were prepared by electrospinning a chitosan/AcOH solution. Encapsulation of the antigen inside the chitosan nanofibers was confirmed through infrared spectroscopy analysis [FTIR]. Guinea pigs were immunized with free antigen and CtxB antigen or antigen alone by direct administration of antigencontaining chitosan nanofibers into the buccal cavity. Serum immunoglobulin G [IgG] and intestinal immunoglobulin A [IgA] antibody responses were determined

Results: The results indicated that antigen in the chitosan nanofibers or nanocapsules elicited very high IgA and IgG responses. No detectable IgA and IgG responses were obtained after oral immunization with CtxB. The results of the antibody titer were analyzed using the ANOVA and LSD tests

Conclusion: CtxB inside the nanofiber increased antibody production when administered orally. This system might be used for delivery of other antigens

Immunogenicity of a new recombinant IpaC from shigella dysenteriae type I in guinea pig as a vaccine candidate

Recombinant vaccine technology is one of the most developed means in controlling infectious diseases. However, an effective vaccine against Shigella is still missing. To evaluate recombinant IpaC protein of Shigella as a vaccine candidate. In this study we cloned IpaC gene into an expression vector in prokaryotic system. The protein expression was evaluated by SDS-PAGE and Western-Blotting analysis. The recombinant protein was purified using Ni-NTA affinity chromatography. Guinea pigs were immunized with the recombinant protein and the level of immunogenicity was examined by ELISA and Western blotting of IpaC. Challenge test was done through the intraoculary injection of Shigella dysenteriae [6×108 CFU/eye] and after 48 hours was scored for keratoconjunctivitis. The results showed a remarkable level of immunogenicity in terms of antibody response and protection against keratoconjunctivitis in tested animals. The recombinant IpaC protein provided a protective system against Shigella dysenteriae type I during the challenge test. The results showed the potential of using recombinant IpaC in preparation of vaccine in perspective studies

[Cloning virG gene and generation mutant construct of pGEM delta virG in order to induction recombination in native Shigella dysenteriae]

Shigellosis disease is the major causes of morbidity in children with diarrhea in Iran. The virG [icsA] gene plays a key role in pathogenesis and ability of invasion in shigella. The aim of this study was cloning, sequencing virG gene and developing a mutant construct pGEM delta virG in order to induction recombination in a native shigella for generation a live attenuated vaccine candidate strain. Initially, by use of biochemical tests, the native shigella strain was detected. The virG gene was cloned in pGEM-7zf vector and the nucleotide sequence was determined. According to the data of sequencing, digestion mapping of pGEMvirG vactor was obtained and a part of virG gene by using enzymatic digestion was removed. Finally, pGEM delta virG construct was transformed to E. coli by utilization of chemical transformation method. The native shigella strain by using biochemical tests was confirmed. The result of sequencing virG gene [native strain] was submitted in NCBI Genebank database. The pGEM delta virG construct contains a mutant construct of virG gene which 1751 bp was deleted through enzymatic digestion reaction and transformed in E. coli. Using the technique of allelic exchange based on the incident of recombination in bacteria is one of the most effective methods to develop a disruption in the target genes. This mutant construct can be applied in development of a live attenuated Shigella dysenteriae vaccine candidate.

Detection of sea, seb, sec, seq genes in staphylococcus aureus isolated from nasal carriers in Tehran province, Iran; by multiplex PCR

Staphylococcus[S.] aureus produces different extra-cellular protein toxins and virulence factors. One of the most important extra-cellular proteins is an enterotoxin which causes staphylococcal food poisoning [SFP] due to their enterotoxins. Different methods have been used to detect this toxin, each of which has advantages and disadvantages. DNA amplification methods, however, can show the presence of enterotoxigenic strains of S. aureus before the expression of enterotoxins on the basis of specific gene sequences. In this study, 150 S. aureus strains isolated from nasal carriers were confirmed by biochemical testing. PCR was used to amplify the staphylococcal enterotoxin A, B, C and Q genes, as well as the staphylococcal nuclease gene. Among the 150 healthy human isolates from the nasal carrier, 95 were confirmed as S. aureus. Only 58.9% of the isolates were diagnosed as sea, b, c, q positive. There were 24 [25.3%] isolates associated with the sea gene, 15.8% isolates associated with the seb gene, 9.5% of the isolates were associated with the sec gene, and 8.4% of the isolates associated with the seq gene. Of these isolates, 41% might be possessing additional se genes but they were not see [178 bp] and sed [319 bp] genes. The nuc gene, which encodes thermo nuclease, was used as a target DNA to identify S. aureus. Additionally, one of these enterotoxigenic isolates carried more than one toxin gene

Cloning and Expression of N-terminal Region of IpaD from Shigella dysenteriae in E. coli

Genus Shigella is one of the important members of the family Enterobacteriaceae. There are numerous antigens in Shigella carrying by a 220 kb plasmid. Among them, IpaD is the key virulence factor of S. flexneri. Apart from having effectors function that is essential for host cell invasion and intracellular survival, this protein also controls the secretion and translocation of other effector proteins into eukaryotic host cells. In the present study, we have cloned and expressed the ipaD in E. coli. The ipaD gene was amplified by PCR. Prokaryote expression vector pET-28a [+] - ipaD was constructed, and used to transform E. coli BL21DE3 plySs. The expression of recombinant protein induced by IPTG was examined by SDS-PAGE. Western blot were used to determine immunoreactivity of IpaD-His by a rabbit monoclonal antibodies against his-tag. SDS-PAGE demonstrated that the constructed prokaryotic expression efficiently produced IpaD at the 1 mmol/L of IPTG. IpaD protein was able to react with the rabbit monoclonal antibody against His-tag. IpaD is essential for Shigella spp invasion. N-terminal region is most significant functional fragment of IpaD. Purification of IpaD from the wild type of Shigella is difficult furthermore profound study on a specific domain on the N-terminal of IpaD by using the wild type of purified IpaD is not feasible

enhancement of biodesulfurization activity in a novel indigenous engineered pseudomonas putida

The combustion of sulfur-rich fossil fuels leads to release of sulfur oxide pollution in the environment. In biodesulfurization process, an organism is able to remove sulfur from fossil fuels without decreasing the caloric value of those substrates. The main aim of this research was to design a recombinant microorganism to remove the highest amount of sulfur compounds in fossil fuels. Three genes [dszA,B,C] from dsz operon are responsible for the 4S pathway [biodesulfurization pathway] in Rhodococcus erythropolis IGTS8 were inserted into the chromosome of a novel indigenous Pseudomonas putida. The reaction catalyzed by products of dszA,B,C genes require FMNH2 supplied by dszD enzyme. Thus, pVLT31 vector harboring dszD gene was transferred into this recombinant strain. The results demonstrated a higher biodesulfurization activity when the flavin reductase gene was transferred into recombinant P. putida harboring dszA,B,C. These results were approved by the Gibbs test and HPLC analysis. These analyses showed that this novel indigenous engineered P. putida could be a promising candidate for an industrial and environmental application for Biodesulfurization process

Simultaneous molecular detection of Neisseria Meningitidis and Haemophilus Influenzae

Bacterial meningitis is a dangerous and sometimes fatal infection that affects the central nervous system. Because some antibiotics can prevent some types of these Bacteria and suppress them from spreading and infecting, therefore it is important to know what type of virus or bacterium is causing meningitis. Haemophilus influenzae and Neisseria meningitides are the two main pathogens causing acute bacterial meningitis. Different methods are used for the detection of H. influenzae and N. meningitidis but they are of low sensitivity, taking long time and difficult to perform . Therefore, complementary methods are necessary for more sensitive detection of these agents. In this study, a muliplex polymerase chain reation [mPCR] assay was developed for detection of H. influenzae and N. meningitidis. These strains were confirmed by biochemical methods. Two specific primer pairs were designed for lic-1 and opa genes of H. influenzae and N. meningitidis respectively. DNA amplification product fragments were 150 bp and 320 bp for H. influenzae and N. meningitidis, respectively. Streptococcus pneumoniae used as a negative control and did not yield a PCR product. The result of this study indicated that PCR is a useful complementary diagnostic technique, especially when Gram stain, culture, or antigenic detection is negative or inconclusive

[Detection of staphylococcus aureus enterotoxin type C isolated from healthy carriers by PCR]

Staphylococcus aureus is a pathogen which can cause food poisoning under certain conditions though growth in nutrients and producing enterotoxin. Only some strains are capable of producing enterotoxin and causing food poisoning and their presence can be detected by DNA amplification and gene sequence specification. Therefore, this research was conducted to detect type C enterotoxinproducing staphylococcus aureus. 95 staphylococcus aureus strains were isolated from 150 nasal carriers using sterilized swabs and were confirmed by biochemical tests. Then primers were designed and the PCR was used to amplify amplify the staphylococcal enterotoxin C gene [sec] in order to detect type C enterotoxogenic strains. DNA amplification fragments of 397 bp for staphylococcal nuclease and those of 271 bp for type C gene were confirmed by enzymatic digestion. Only 9.5% of the isolated strains contained sec gene. Specificity and sensitivity were also evaluated and its sensitivity was found to be 125 cells. This technique is a rapid, sensitive, specific, inexpensive and different alternative to conventional biochemical and serologic assays and it can be used to detect the agent producing type C staphylococcal enterotoxin