Effect of C-Terminus Modification in Salmonella typhimurium FliC on Protein Purification Efficacy and Bioactivity.

Recombinant flagellin (FliC) has shown low efficacy in purification because of inclusion bodies formation and aggregation. We hypothesized preserving TLR5 binding site of FliC and removing some amino acids could be responsible for aggregation and solubility improvement. Hence, a bioinformatics study was performed to find hotspots in aggregate formation. Protein modeling was carried out by SWISS-MODEL and I-TASSER servers and models were compared by MATRAS server and Chimera 1.11.2. Gene modification was carried out based on bioinformatics studies. Genes, (truncated modified fliC (tmFliC) and full-length fliC (flFliC)), were cloned and expressed in pET-21a vector. Protein purification was carried out using HIS-Tag method. Proliferation assay and also induction of IL-8 in HEK293 cells were performed to confirm bioactivity function of tmFliC. Bioinformatics results showed that partial deletion of C-terminus may increase solubility without unfavorable effect on TLR5 recognition. Also, model comparison showed that this protein may preserve 3D structure. In addition, GlobPlot server demonstrated that tmFliC formed its globular domains which were important in TLR5 recognition. As we expected, high purification efficacy for tmFliC compared with flFliC was also obtained in experimental studies and a proper function for tmFliC was observed. The tmFliC enhanced cell proliferation in HEK293 cells compare with control after 24 h. Also, IL-8 level was increased with stimulation by tmFliC after 24 h. In conclusion, reducing hydrophobicity in C-terminus and deleting necessary amino acids for filament formation may increase protein solubility.

"In-house" production of DNA size marker from a vaccinal Bacillus anthracis strain.

BACKGROUND AND OBJECTIVES: DNA molecular weight marker is widely used in molecular biology experiments incurring considerable costs on low-budget settings. MATERIALS AND METHODS: Here a PCR-supported procedure is described that uses 10 primer pairs targeting chromosomal DNA from the harmless vaccinal Bacillus anthracis Sterne 34F2 strain as template. A single PCR protocol is used to reproduce all the 10 fragments of a 100 bp DNA size marker. RESULTS AND CONCLUSION: The unpurified amalgam of 10 PCR products can be directly loaded to agarose gels. This work was intended to develop a reasonably cost-effective DNA ladder that is useful for researchers in laboratories with limited funding.

Vaccination with recombinant 4 × M2e.HSP70c fusion protein as a universal vaccine candidate enhances both humoral and cell-mediated immune responses and decreases viral shedding against experimental challenge of H9N2 influenza in chickens.

As cellular immunity is essential for virus clearance, it is commonly accepted that no adequate cellular immunity is achieved by all available inactivated HA-based influenza vaccines. Thus, an improved influenza vaccine to induce both humoral and cell-mediated immune responses is urgently required to control LPAI H9N2 outbreaks in poultry farms. M2e-based vaccines have been suggested and developed as a new generation of universal vaccine candidate against influenza A infection. Our previous study have shown that a prime-boost administration of recombinant 4×M2e.HSP70c (r4M2e/H70c) fusion protein compared to conventional HA-based influenza vaccines provided full protection against lethal dose of influenza A viruses in mice. In the present study, the immunogenicity and protective efficacy of (r4M2e/H70c) was examined in chickens. The data reported herein show that protection against H9N2 viral challenge was significantly increased in chickens by injection of r4M2e/H70c compared with injection of conventional HA-based influenza vaccine adjuvanted with MF59 or recombinant 4×M2e (r4M2e) without HSP70c. Oropharyngeal and cloacal shedding of the virus was detected in all of the r4M2e/H70c vaccinated birds at 2 days after challenge, but the titer was low and decreased rapidly to reach undetectable levels at 7 days after challenge. Moreover, comparison of protective efficacy against LPAI H9N2 in birds intramuscularly immunized with r4M2e/H70c likely represented the ability of the M2e-based vaccine in providing cross-protection against heterosubtypic H9N2 challenge and also allowed the host immune system to induce HA-homosubtype neutralizing antibody against H9N2 challenge. This protective immunity might be attributed to enhanced cell-mediated immunity, which is interpreted as increased lymphocytes proliferation, increased levels of Th1-type (IFN-γ) and Th2-type (IL-4) cytokines production and increased CD4(+) to CD8(+) ratios, resulting from the injection of four tandem repeats of the ectodomain of the conserved influenza matrix protein M2 (4×M2e) genetically fused to C-terminus of Mycobacterium tuberculosis HSP70 (mHSP70c).

The population structure of Salmonella enterica Enteritidis in Iran analyzed by multiple-locus variable-number tandem repeat analysis.

Salmonella enterica Enteritidis is the most frequent etiological agent of salmonellosis in humans and poultry. To understand the genetic diversity of S. Enteritidis in Iran, we examined 69 chicken isolates from 18 broiler farms and six non-epidemic human isolates from six geographically distant provinces by multi-locus variable-number tandem repeat analysis (MLVA). Among SE2, SE3, SE5, SE7, SE8, SENTR4, and SENTR7, only SE5 with four and SENTR7 with two alleles, respectively, proved variable giving estimates of locus genetic diversity of 0.58 and 0. In all, six closely related MLVA profiles were identified among which three were commonly represented by human and chicken isolates. This population homogeneity contrasts with the high diversity at these loci reported elsewhere and is likely a consequence of a single clone of S. Enteritidis distributed across Iran.

The phenotypic and genotypic characterization of Bacillus anthracis isolates from Iran.

To understand epidemiology of Bacillus anthracis in Iran, the morphological, biochemical, and virulence specifications of 32 B. anthracis isolates, collected from human, sheep, cattle, goat, and environmental specimens obtained from throughout Iran were examined by conventional and molecular approaches. B. anthracis isolates were characterized in multiple ways: (1) capsule formation both on bicarbonate agar and in defibrinated horse blood, (2) motility of vegetative forms, (3) hemolysis on 5% sheep blood agar, (4) penicillin G susceptibility, (5) lecithinase production on egg yolk agar, (6) gelatin hydrolysis, (7) ability to develop "string of pearls" on tryptose agar, and (8) capability to develop mucoid colonies in presence of CO(2) were assessed. In addition, biochemical properties such as indole, methyl red, catalase, citrate utilization, and finally nitrate reduction tests were used. All the tested isolates produced identical morphological and biochemical patterns with those of the vaccine strain B. anthracis 34F2 Sterne. In order to assess potential virulence of isolates at genomic level, PCR protocols assaying for the pXO1 and pXO2 loci were employed. The intriguing high level of phenotypic similarity between Iranian isolates of B. anthracis and the 34F2 Sterne strain deserves further studies at genomic level.