Interaction of camel Lactoferrin derived peptides with DNA: a molecular dynamics study.

BACKGROUND: Lactoferrampin (LFampin), Lactoferricin (LFcin), and LFchimera are three well-known antimicrobial peptides derived from Lactoferrin and proposed as alternatives for antibiotics. Although the intracellular activity of these peptides has been previously demonstrated, their mode of action is not yet fully understood. Here, we performed a molecular dynamics simulation study to understand the molecular interactions between camel Lactoferrin derived peptides, including CLFampin, CLFcin, and CLFchimera, and DNA as an important intracellular target. RESULTS: Our results indicate that all three peptides bind to DNA, albeit with different propensities, with CLFchimera showing the highest binding affinity. The secondary structures of the peptides, modeled on Lactoferrin, did not undergo significant changes during simulation, supporting their functional relevance. Main residues involved in the peptide-DNA interaction were identified based on binding free energy estimates calculated over 200 ns, which, as expected, confirmed strong electrostatic interactions between DNA phosphate groups and positively charged peptide side chains. Interaction between the different concentrations of CLFchimera and DNA revealed that after binding of four copies of CLFchimera to DNA, hydrogen bonds between the two strands of DNA start to break from one of the termini. CONCLUSIONS: Importantly, our results revealed that there is no DNA-sequence preference for peptide binding, in line with a broad antimicrobial activity. Moreover, the results showed that the strength of the interaction between DNA and CLFchimera is concentration dependent. The insight provided by these results can be used for the rational redesign of natural antimicrobial peptides targeting the bacterial DNA.

Immunogenic evaluation of FMD virus immuno-dominant epitopes coupled with IL-2/FcIgG in BALB/c mice.

Previous studies on vaccine development against foot-and-mouth disease (FMD) virus reported that application of the inactivated vaccines for FMD virus is not completely effective. Novel vaccinations based on immune-dominant epitopes showed they induced immune responses. In addition, for better and safer immunization, access to of efficient adjuvants against FMD virus seems to be critical. In this study, we produced epitope recombinant vaccines from the VP1 protein of the FMD virus for serotype O of Iran that conjugated with Fc Immunoglobulin (FcIgG) and Interleukin-2 (IL-2). Multiple-epitope constructs included Polytope, Polytope-IL2-FcIgG, Polytope-IL2, Polytope-FcIgG that cloned and expressed in E. coli BL21 (DE3). To evaluate whether these epitope recombinant vaccines induce immune responses, BALB/c mice were injected with the epitope recombinant vaccines and their immune responses were compared with a negative control group. The humoral and cellular immune responses were measured by ELISA. The results showed there were significant differences between the negative control group and other immunized mice with recombinant epitope proteins (p < 0.05). The results of total IgG, IgG1, IgG2a levels and secretion of IFN-γ, IL-4 and IL-10 revealed that immune responses were enhanced when the epitope recombinant vaccine of FMD virus coupled with IL-2 and FcIgG. Observations indicated that the epitope recombinant plasmid of the VP1 protein co-expressed with IL-2 and FcIgG was effective in inducing an enhanced immune response. Therefore, IL-2 and FcIgG could be recommended as a potential adjuvant for epitope recombinant vaccine of the VP1 protein from FMD virus.

In vivo immunogenicity assessment and vaccine efficacy evaluation of a chimeric tandem repeat of epitopic region of OMP31 antigen fused to interleukin 2 (IL-2) against Brucella melitensis in BALB/c mice.

BACKGROUND: Designing a potent recombinant vaccine, using the appropriate subunits with the greatest effect on stimulating the immune system, especially in the case of intracellular pathogens such as gram negative Brucella Melitensis bacteria, is of great importance. In this study, three repeats of 27 amino acids of the immunogenic epitope derived from OMP31 antigen (3E) from the Brucella melitensis, in a protective manner against Brucellosis have been used. To fortify the delivery system of recombinant antigens, IL-2 cytokine as a molecular adjuvant was fused to recombinant constructs. Recombinant proteins were evaluated for immunological studies in a mouse model (BALB/c). RESULTS: The results showed that all recombinant proteins could stimulate the immune system to produce Th1 cytokines and antibodies in compare to the negative control treatments. 3E-IL2 and then OMP31-IL2 proteins stimulated higher levels of IFN-γ and IL-2 compared to the other treatments (p < 0.05). Also, the results indicated that experimental treatments produced a higher level of IgG2a isotype than IgG1 isotype. In addition, the findings of the experiment showed that the presence of chemical adjuvant (IFA) along with molecular adjuvant can play a significant role in stimulating the immune system. After determining the potency of recombinant structures, their efficacy in stimulating the immune system were also evaluated. B. melitensis M16 strain was used to challenge 30 days after last immunization. The microbial load of the splenocyte in the treatments receiving chimeric proteins were significantly lower. Also, Wright serological test confirmed that these treatments had the lowest agglutination rate, as well as the positive treatment, while in the negative treatments in excess of blood serum dilutions, agglutination rate were more than 2 + . CONCLUSIONS: 3E-IL2 treatment showed the best performance compared to other recombinant proteins and could be considered as the suitable candidate for further research on the production of recombinant vaccine against Brucella.

Evaluation of immune responses induced by polymeric OMP25-BLS Brucella antigen.

Brucellosis is one the serious infectious diseases caused deleterious health and economic losses. Vaccination with subunit vaccines is the efficient alternative way than live attenuated vaccines against infectious diseases. Herein a new chimeric OMP25-BLS antigen emulsified in Chitosan Nanoparticles was designed and its immune responses were compared with control groups. Also, the role of heat shock protein 60 kDa in combination with OMP25-BLS antigen was assessed. Structural and antigenic features of chimeric antigen were predicted using bioinformatics tools. Moreover, the humoral and cellular immune responses were measured by ELISA in seven different groups. Observations showed rOMP25-BLS structure was highly stable and antigenic. Cytokines analysis showed rOMP25 and rOMP25-BLS + rHSP60 induced higher titer of INF-γ than rHSP60 and rOMP25-BLS. There was no statistically significant difference between positive control group and rOMP25-BLS + rHSP60 in inducing TNF-α (p < .05). Additionally, the highest titer of IL-4 was dedicated to rOMP25 among other immunized treatments, while there were no significant differences between positive control group and other immunized groups with recombinant proteins (p < .05). In addition, rOMP25-BLS and rHSP60 induced higher titer of total antibody compared to other groups. Also, rHSP60 could improve IgG2a to IgG1 ratio when it used in combination with chimeric antigen. Moreover, the lymphocyte proliferation index was higher in chimeric rOMP25-BLS + HSP60 antigen. In conclusion, while rOMP25-BLS chimeric antigen unable to induce efficient cellular response than individual injection of rOMP25, its injection in combination with rHSP60 could improve cellular immunity.

Paternal breed effects on expression of IGF-II, BAK1 and BCL2-L1 in bovine preimplantation embryos.

The effects of the paternal breed on early embryo and later pre- and postnatal development are well documented. Several recent studies have suggested that such paternal effects may be mediated by the paternally induced epigenetic modifications during early embryogenesis. The objective of this study was to investigate the effects of the paternal breed on the early embryonic development and relative expression of the maternally imprinted gene, IGF-II, and the apoptosis-related genes BAK1 and BCL2-L1 in in vitro produced (IVP) bovine embryos derived from two unrelated paternal breeds (Holstein and Brown Swiss). The degree of correlation of IGF-II expression pattern with embryo developmental competence and apoptosis-related genes was also investigated. The relative abundance of IGF-II, BCL2-L1 and BAK1 transcripts in day 8 embryos was measured by quantitative reverse-transcription polymerase chain reaction using the comparative Cp method. Our data revealed that the paternal breed did not influence cleavage rate, blastocyst rate and relative abundance of IGF-II, BAK1 and BCL2-L1 in day 8 blastocysts (P > 0.05). Nevertheless, IGF-II expression levels were highly correlated with embryonic developmental competence (r = 0.66, P < 0.1), relative expression of BCL2-L1 (r = 0.72, P < 0.05) and ratio of BCL2-L1/BAK1 (r = 0.78, P < 0.05). In conclusion, our data show that IGF-II, BCL2-L1 and BAK1 expression is not related to the chosen combination of paternal breed, but that IGF-II expression is correlated with embryonic viability and apoptosis-related gene expression.

Engineering Human Pancreatic RNase 1 as an Immunotherapeutic Agent for Cancer Therapy Through Computational and Experimental Studies.

Most plant and bacterial toxins are highly immunogenic with non-specific toxic effects. Human ribonucleases are thought to provide a promising basis for reducing the toxic agent's immunogenic properties, which are candidates for cancer therapy. In the cell, the ribonuclease inhibitor (RI) protein binds to the ribonuclease enzyme and forms a tight complex. This study aimed to engineer and provide a gene construct encoding an improved version of Human Pancreatic RNase 1 (HP-RNase 1) to reduce connection to RI and modulate the immunogenic effects of immunotoxins. To further characterize the interaction complex of HP-RNase 1 and RI, we established various in silico and in vitro approaches. These methods allowed us to specifically monitor interactions within native and engineered HP-RNase 1/RI complexes. In silico research involved molecular dynamics (MD) simulations of native and mutant HP-RNase 1 in their free form and when bound to RI. For HP-RNase 1 engineering, we designed five mutations (K8A/N72A/N89A/R92D/E112/A) based on literature studies, as this combination proved effective for the intended investigation. Then, the cDNA encoding HP-RNase 1 was generated by RT-PCR from blood and cloned into the pSYN2 expression vector. Consequently, wild-type and the engineered HP-RNase 1 were over-expressed in E. coli TG1 and purified using an IMAC column directed against a poly-his tag. The protein products were detected by SDS-PAGE and Western blot analysis. HP-RNase 1 catalytic activity, in the presence of various concentrations of RI, demonstrated that the mutated version of the protein is able to escape the ribonuclease inhibitor and target the RNA substrate 2.5 folds more than that of the wild type. From these data, we tend to suggest the engineered recombinant HP-RNase 1 potentially as a new immunotherapeutic agent for application in human cancer therapy.

Drug repositioning for immunotherapy in breast cancer using single-cell analysis.

Immunomodulatory peptides, while exhibiting potential antimicrobial, antifungal, and/or antiviral properties, can play a role in stimulating or suppressing the immune system, especially in pathological conditions like breast cancer (BC). Thus, deregulation of these peptides may serve as an immunotherapeutic strategy to enhance the immune response. In this meta-analysis, we utilized single-cell RNA sequencing data and known therapeutic peptides to investigate the deregulation of these peptides in malignant versus normal human breast epithelial cells. We corroborated our findings at the chromatin level using ATAC-seq. Additionally, we assessed the protein levels in various BC cell lines. Moreover, our in-house drug repositioning approach was employed to identify potential drugs that could positively impact the relapse-free survival of BC patients. Considering significantly deregulated therapeutic peptides and their role in BC pathology, our approach aims to downregulate B2M and SLPI, while upregulating PIGR, DEFB1, LTF, CLU, S100A7, and SCGB2A1 in BC epithelial cells through our drug repositioning pipeline. Leveraging the LINCS L1000 database, we propose BRD-A06641369 for B2M downregulation and ST-4070043 and BRD-K97926541 for SLPI downregulation without negatively affecting the MHC complex as a significantly correlated pathway with these two genes. Furthermore, we have compiled a comprehensive list of drugs for the upregulation of other selected immunomodulatory peptides. Employing an immunotherapeutic approach by integrating our drug repositioning pipeline with single-cell analysis, we proposed potential drugs and drug targets to fortify the immune system against BC.

The immunomodulatory effects of lactoferrin and its derived peptides on NF-κB signaling pathway: A systematic review and meta-analysis.

BACKGROUND: Lactoferrin is a versatile protein with important modulatory functions in inflammation and immune response. This glycoprotein can bind and sequester iron and LPS, thereby intervening in certain signaling pathways and biological processes. In the present meta-analysis, we aimed to pool experimental data regarding the immunomodulatory effects of lactoferrin and its derived peptides on the NF-κB signaling pathway. MATERIALS: We searched PubMed, Google Scholar, and Web of Science databases and obtained all related articles published before April 2022. Finally, 25 eligible studies were selected, and their reports were analyzed. METHODS: We used Review Manager Version 5.2 to compute the standardized mean difference (SMD) and its 95% confidence interval. In addition, the source of heterogeneity was explored using meta-regression and sensitivity analysis. The symmetry of the funnel plot and Egger's test were also used to evaluate publication bias utilizing Comprehensive Meta-Analysis Version 2. RESULTS: Comparing the group of cells and animals exposed to lipopolysaccharide alone with the group that received pretreatment with lactoferrin and its derivatives, we observed significant reductions in TNF-α, IL-1 beta, and IL-6 levels by 8.73 pg/mL, 2.21 pg/mL, and 3.24 pg/mL, respectively, in the second group. Additionally, IKK-ß, p-IκB, and NF-κB (p65) levels were significantly lower by 7.37-fold, 15.02-fold, and 3.88-fold, respectively, in various cells and tissues. CONCLUSION: Based on the results of this meta-analysis, lactoferrin and its derived peptides can be considered potent prophylactic and therapeutic candidates against inflammation-associated diseases by targeting the NF-kB pathway.

Improvement of the performance of anticancer peptides using a drug repositioning pipeline.

The use of anticancer peptides (ACPs) as an alternative/complementary strategy to conventional chemotherapy treatments has been shown to decrease drug resistance and/or severe side effects. However, the efficacy of the positively-charged ACP is inhibited by elevated levels of negatively-charged cell-surface components which trap the peptides and prevent their contact with the cell membrane. Consequently, this decreases ACP-mediated membrane pore formation and cell lysis. Negatively-charged heparan sulphate (HS) and chondroitin sulphate (CS) have been shown to inhibit the cytotoxic effect of ACPs. In this study, we propose a strategy to promote the broad utilization of ACPs. In this context, we developed a drug repositioning pipeline to analyse transcriptomics data generated for four different cancer cell lines (A549, HEPG2, HT29, and MCF7) treated with hundreds of drugs in the LINCS L1000 project. Based on previous studies identifying genes modulating levels of the glycosaminoglycans (GAGs) HS and CS at the cell surface, our analysis aimed at identifying drugs inhibiting genes correlated with high HS and CS levels. As a result, we identified six chemicals as likely repositionable drugs with the potential to enhance the performance of ACPs. The codes in R and Python programming languages are publicly available in https://github.com/ElyasMo/ACPs_HS_HSPGs_CS. As a conclusion, these six drugs are highlighted as excellent targets for synergistic studies with ACPs aimed at lowering the costs associated with ACP-treatment.

PCR-SSCP variation of GH and STAT5A genes and their association with estimated breeding values of growth traits in Baluchi sheep.

Growth hormone (GH) selected for its important role in economically relevant traits and signal transducers and activators of transcription 5A (STAT5A) is also known as a main mediator of growth hormone action on target genes. A total number of 190 lambs of Iranian purebred Baluchi sheep were genotyped at exon 5 of GH and exons 7 and 8 of STAT5A genes by using PCR-SSCP analysis. GH gene revealed three (G1, G2, and G3) conformational patterns; however, STAT5A loci were not polymorphic. Breeding values of growth traits including birth weight, weaning weight, 6 months weight, 9 months weight, and yearling weight were estimated by using the Best Linear Unbiased Prediction based on an animal model with a relationship matrix. Studied growth traits were examined for association analysis. Our findings suggest that animals with G2 genotype have highest breeding value for six month weight, while these animals have lowest breeding value for pre-weaning traits. Higher performance of G2 animals in adult ages may be related to the growth hormone role in puberty ages. The other traits showed no relationship to the genotypes examined.

Nanoparticle or conventional adjuvants: which one improves immune response against Brucellosis?

OBJECTIVES: Brucellosis is a common infectious disease among animals and humans. While subunit vaccines could be used as an efficient strategy against pathogens, they usually seem to be less immunogenic than live or killed vaccines. However, the use of a suitable adjuvant accompanied by subunit vaccines can be a good alternative to enhance the immune response. MATERIALS AND METHODS: To find a proper adjuvant against Brucellosis, the immune response of induced mice by Aluminum Hydroxide (AH), Incomplete Freund (IFA), and Chitosan Nanoparticle (CS) adjuvants in individuals and in combination with CS were assessed. RESULTS: Immunization with CS stimulated higher interferon gamma (IFN-γ) immunity, while there were no significant differences between rOMP25 (IFA), rOMP25 (AH), rOMP25 (AH-CS) and rOMP25 (IFA-CS) recombinant proteins. Tumor necrosis factor alpha (TNF-α) analysis revealed there were no significant differencesbetween immunized groups and the positive control group, except for the treatment formulated in single IFA. Furthermore, unlike IFN-γ, there was a reverse interleukin-4 (IL-4) immune response trend for treatments, as rOMP25 (CS) displayed the lowest response. rOMP25 (CS) induced higher titer of total antibody than the other ones. Although the recombinant proteins emulsified in different adjuvants induced similar titer of IgG1 antibody, the ones that were formulated in CS, IFA and IFA-CS showed a higher titer of IgG2a. The cell proliferation assay demonstrating the antigen-specific cell proliferative response could be promoted after immunization with CS. CONCLUSION: CS whether single or in combination with IF adjuvants has potential to improve Th1-Th2 responses.

Effect of IL-2 co-expressed or co-inoculated with immuno-dominant epitopes from VP1 protein of FMD virus on immune responses in BALB/c mice.

OBJECTIVES: The results of studies on vaccine development for foot-and-mouth disease (FMD) virus show that the use of inactivated vaccines for FMD virus is not completely effective. Novel vaccinations based on immuno-dominant epitopes have been shown to induce immune responses. Furthermore, for safety of immunization, access to efficient adjuvants against FMD virus seems to be critical. MATERIALS AND METHODS: In this study, we produced epitope recombinant vaccines from the VP1 protein of the FMD virus for serotype O of Iran. Constructs were included polytope (tandem-repeat multiple-epitope), polytope coupled with interleukin-2 (polytope-IL 2) as a molecular adjuvant and IL-2. Three expression vectors were constructed and expressed in Escherichia coli BL21 (DE3). To evaluate whether these recombinant vaccines induce immune responses, BALB/c mice were injected with the recombinant vaccines and their immune responses were compared with a negative control group. The humoral and cellular immune responses were measured by ELISA. RESULTS: The results showed that IL-2 co-expressed or co-inoculated with Polytope protein enhances the immune effect of multiple epitope recombinant vaccine against FMD virus. The results of total immunoglobulin G (IgG), IgG1, and IgG2a levels and secretion of interferon gamma (IFN-γ), IL-4 and IL-10 revealed that there were significant differences between negative control group and other injected mice with the recombinant vaccines (P<0.05). CONCLUSION: Observations indicated that the epitope recombinant plasmid of the VP1 protein co-expressed or co-inoculated with IL-2 was effective in inducing an enhanced immune response. Therefore, IL-2 can be recommended as a potential adjuvant for epitope recombinant vaccine of the VP1 protein from FMD virus.

Dynamics of The Expression of Pluripotency and Lineage Specific Genes in The Pre and Peri-Implantation Goat Embryo.

OBJECTIVE: Two critical points of early development are the first and second lineage segregations, which are regulated by a wide spectrum of molecular and cellular factors. Gene regulatory networks, are one of the important components which handle inner cell mass (ICM) and trophectoderm (TE) fates and the pluripotency status across different mammalian species. Considering the importance of goats in agriculture and biotechnology, this study set out to investigate the dynamics of expression of the core pluripotency markers at the mRNA and protein levels. MATERIALS AND METHODS: In this experimental study, the expression pattern of three pluripotency markers (Oct4, Nanog and Sox2) and the linage specific markers (Rex1, Gata4 and Cdx2) were quantitatively assessed in in vitro matured (MII) oocytes and embryos at three distinctive stages: 8-16 cell stage, day-7 (D7) blastocysts and D14 blastocysts. Moreover, expression of Nanog, Oct4, Sox2 proteins, and their localization in the goat blastocyst was observed through immunocytochemistry. RESULTS: Relative levels of mRNA transcripts for Nanog and Sox2 in D3 (8-16 cell) embryos were significantly higher than D7 blastocysts and mature oocytes, while Oct4 was only significantly higher than D7 blastocysts. However, the expression pattern of Rex1, as an epiblast linage marker, decreased from the oocyte to the D14 stage. The expression pattern of Gata4 and Cdx2, as extra embryonic linage markers, also showed a similar trend from oocyte to D3 while their expressions were up-regulated in D14 blastocysts. CONCLUSION: Reduction in Nanog, Oct4, Sox2 mRNA transcription and a late increase in extra embryonic linage markers suggests that the developmental program of linage differentiation is retarded in goat embryos compared to previously reported data on mice and humans. This is likely related to late the implantation in goats.

Immunogenicity evaluation of plasmids encoding Brucella melitensis Omp25 and Omp31 antigens in BALB/c mice.

OBJECTIVES: Vaccination is one of the most effective means to protect humans and animals against brucellosis. Live attenuated Brucella vaccines are considered effective in animals but they may be potentially infectious to humans, so it is vital to improve the immunoprotective effects and safety of vaccines against Brucella. This study was designed to evaluate the immunogenicity of DNA vaccines encoding B. melitensis outer membrane proteins (Omp25 and Omp31) against B. melitensis Rev1 in a mouse model. MATERIALS AND METHODS: For this propose, Omp25 and Omp31 genes were cloned (individually and together) into the eukaryotic expression vector pcDNA3.1/Hygro (+). Expressions of recombinant plasmids were confirmed by SDS-PAGE and Western blot analysis. Six groups of BALB/c mice (seven mice per group) were intramuscularly injected with three recombinant constructs, native pcDNA3.1/Hygro (+) and phosphate-buffered saline (PBS) as controls and subcutaneous injection of attenuated live vaccine Rev1. RESULTS: Results indicated that DNA vaccine immunized BALB/c mice had a dominant immunoglobulin G response and elicited a T-cell-proliferative response and induced significant levels of interferon gamma (INF-γ) compared to the control groups. CONCLUSION: Collectively, these finding suggested that the pcDNA3.1/Hygro DNA vaccines encoding Omp25 and Omp31 genes and divalent plasmid were able to induce both humoral and cellular immunity, and had the potential to be a vaccine candidate for prevention of B. melitensis infections.

Incorporating Prior Knowledge of Principal Components in Genomic Prediction.

Genomic prediction using a large number of markers is challenging, due to the curse of dimensionality as well as multicollinearity arising from linkage disequilibrium between markers. Several methods have been proposed to solve these problems such as Principal Component Analysis (PCA) that is commonly used to reduce the dimension of predictor variables by generating orthogonal variables. Usually, the knowledge from PCA is incorporated in genomic prediction, assuming equal variance for the PCs or a variance proportional to the eigenvalues, both treat variances as fixed. Here, three prior distributions including normal, scaled-t and double exponential were assumed for PC effects in a Bayesian framework with a subset of PCs. These developed PCR models (dPCRm) were compared to routine genomic prediction models (RGPM) i.e., ridge and Bayesian ridge regression, BayesA, BayesB, and PC regression with a subset of PCs but PC variances predefined as proportional to the eigenvalues (PCR-Eigen). The performance of methods was compared by simulating a single trait with heritability of 0.25 on a genome consisted of 3,000 SNPs on three chromosomes and QTL numbers of 15, 60, and 105. After 500 generations of random mating as the historical population, a population was isolated and mated for another 15 generations. The generations 8 and 9 of recent population were used as the reference population and the next six generations as validation populations. The accuracy and bias of predictions were evaluated within the reference population, and each of validation populations. The accuracies of dPCRm were similar to RGPM (0.536 to 0.664 vs. 0.542 to 0.671), and higher than the accuracies of PCR-Eigen (0.504 to 0.641) within reference population over different QTL numbers. Decline in accuracies in validation populations were from 0.633 to 0.310, 0.639 to 0.313, and 0.617 to 0.298 using dPCRm, RGPM and PCR-Eigen, respectively. Prediction biases of dPCRm and RGPM were similar and always much less than biases of PCR-Eigen. In conclusion assuming PC variances as random variables via prior specification yielded higher accuracy than PCR-Eigen and same accuracy as RGPM, while fewer predictors were used.

Impact of heat shock protein 60KD in combination with outer membrane proteins on immune response against Brucella melitensis.

Brucellosis caused by the bacterium Brucella affects various domestic and wild species. The outer membrane proteins 25 and 31 play key roles on stimulation of cell-mediated immune response against Brucella. GroEL as one of the major Brucella antigens stimulates the immune system and increases intracellular survival of bacteria. In the present study, we assumed injection of GroEL in combination with OMP25 and OMP31 would offer higher immunity levels. So, the impact of GroEL with different concentrations of recombinant outer membrane proteins emulsified in Chitosan Nanoparticles on immune responses was evaluated in mice model. Results showed both univalent (except rGroEL) and divalent immunized groups induced higher IFN-γ, TNF-α, and IL-4 titers in comparison to negative control groups. While GroEL showed negative effect on TNF-α titer, there were positive increase trends in IFN-γ in some treatments. Analysis of humoral antibody response revealed both univalent and divalent immunized groups induced higher IgG2a titer than IgG1 titer, indicating strong bent of Th1 immune response. Also, results showed GroEL can have positive impact on lymphocyte proliferation response. Overall, mice immunization using individual OMP25 or OMP31 demonstrated more effective cell-mediated immunity, although some combinations of rGroEL and rOMP31 vaccines were more efficient than other divalent ones.

PhiC31-based Site-Specific Transgenesis System for Production of Transgenic Bovine Embryos by Somatic Cell Nuclear Transfer and Intracytoplasmic Sperm Injection.

OBJECTIVES: The Streptomyces phage phiC31 integrase offers a sequence-specific method of transgenesis with a robust long-term gene expression. PhiC31 has been successfully developed in a variety of tissues and organs for purpose of in vivo gene therapy. The objective of the present experiment was to evaluate PhiC31-based site-specific transgenesis system for production of transgenic bovine embryos by somatic cell nuclear transfer and intracytoplasmic sperm injection. MATERIALS AND METHODS: In this experimental study, the application of phiC31 integrase system was evaluated for generating transgenic bovine embryos by somatic cell nuclear transfer (SCNT) and sperm mediated gene transfer (SMGT) approaches. RESULTS: PhiC31 integrase mRNA and protein was produced in vitro and their functionality was confirmed. Seven phiC31 recognizable bovine pseudo attachment sites of phage (attP) sites were considered for evaluation of site specific recombination. The accuracy of these sites was validated in phic31 targeted bovine fibroblasts using polymerase chain reaction (PCR) and sequencing. The efficiency and site-specificity of phiC31 integrase system was also confirmed in generated transgenic bovine embryo which successfully obtained using SCNT and SMGT technique. CONCLUSIONS: The results showed that both SMGT and SCNT-derived embryos were enhanced green fluorescent protein (EGFP) positive and phiC31 integrase could recombine the reporter gene in a site specific manner. These results demonstrate that attP site can be used as a proper location to conduct site directed transgenesis in both mammalian cells and embryos in phiC31 integrase system when even combinaed to SCNT and intracytoplasmic sperm injection (ICSI) method.

Cloning, expression and molecular analysis of Iranian Brucella melitensis Omp25 gene for designing a subunit vaccine.

Brucellosis is a well-known domestic animal infectious disease, which is caused by Brucella bacterium. The outer membrane protein 25 kDa (Omp25) gene plays an important role in simulating of TNF-α, IFN-α, macrophage, and cytokines cells. In the current study molecular cloning and expression analysis of Omp25 gene for designing a subunit vaccine against Brucella was investigated. Amplifying the full length of candidate gene was performed using specific primers. Sub-cloning of this gene conducted using pTZ57R/T vector in TOP10F strain of Escherichia coli(E.coli) as the host. Also, pET32(a)+ vector used for expression in BL21 (DE3) strain of E.coli. Omp25 gene with 642 bp size was amplified and cloned successfully. The expression results were confirmed by sequencing and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analyses which showed 42 kDa protein band correctly. Also, phylogenic analysis showed this gene has a near genetic relation with other Brucella strains. According to our results we can propose this gene as a candidate useful for stimulation of cell-mediated and humoral immunity system in future study.

Design and Construction of Chimeric VP8-S2 Antigen for Bovine Rotavirus and Bovine Coronavirus.

PURPOSE: Bovine Rotavirus and Bovine Coronavirus are the most important causes of diarrhea in newborn calves and in some other species such as pigs and sheep. Rotavirus VP8 subunit is the major determinant of the viral infectivity and neutralization. Spike glycoprotein of coronavirus is responsible for induction of neutralizing antibody response. METHODS: In the present study, several prediction programs were used to predict B and T-cells epitopes, secondary and tertiary structures, antigenicity ability and enzymatic degradation sites. Finally, a chimeric antigen was designed using computational techniques. The chimeric VP8-S2 antigen was constructed. It was cloned and sub-cloned into pGH and pET32a(+) expression vector. The recombinant pET32a(+)-VP8-S2 vector was transferred into E.oli BL21CodonPlus (DE3) as expression host. The recombinant VP8-S2 protein was purified by Ni-NTA chromatography column. RESULTS: The results of colony PCR, enzyme digestion and sequencing showed that the VP8-S2 chimeric antigen has been successfully cloned and sub-cloned into pGH and pET32a(+).The results showed that E.coli was able to express VP8-S2 protein appropriately. This protein was expressed by induction of IPTG at concentration of 1mM and it was confirmed by Ni-NTA column, dot-blotting analysis and SDS-PAGE electrophoresis. CONCLUSION: The results of this study showed that E.coli can be used as an appropriate host to produce the recombinant VP8-S2 protein. This recombinant protein may be suitable to investigate to produce immunoglobulin, recombinant vaccine and diagnostic kit in future studies after it passes biological activity tests in vivo in animal model and or other suitable procedure.

Production of specific IgY antibody to the recombinant FanC protein produced in Escherichia coli.

OBJECTIVES: Enterotoxigenic Escherichia coli (ETEC) strains are one of the primary causes of diarrhea in newborn calves and in humans, pigs, and sheep. IgY technology has been identified as a promising alternative to generating a mass amount of specific antibody for use in immunotherapy and immunodiagnostics. The purpose of this study was to produce specific antibody by egg yolk antibody (IgY) to recombinant FanC protein from ETEC. MATERIALS AND METHODS: FanC (K99) gene was amplified from ETEC by specific primers and polymerase chain reaction. The gene was cloned and subcloned into pTZ57R/T and pET32a (+) vectors, respectively. Recombinant vector was transferred into E. coli BL21 CodonPlus (DE3). Protein expression was investigated by 1 mM IPTG induction. Hens were immunized by the purified recombinant FanC protein. The activity and specificity of the IgY antibody were detected by dot-blotting, Western blotting, and indirect ELISA. RESULTS: We obtained FanC specific IgYs by immunizing the hens with the recombinant FanC protein. The anti-FanC IgY showed binding specifically to the FanC protein of ETEC. CONCLUSION: The results emphasize that specific IgY against the recombinant FanC protein could be recommended as a candidate for passive immunization against ETEC infection in animals and humans.