Characterization and Sequencing of MT-Cox1 Gene in Khorasan Native Poultry

The aim of this study was to investigate the nucleotide sequence of COX1 gene in mitochondrial genome of Khorasan native chicken and detect the possible mutations in the genome. For this purpose, after sampling and extracting DNA from the whole blood samples, the COX1 gene was amplified using specific primers and cloned in pTG19-T plasmid of the DH5α strain of Escherichia coli bacteria. Finally, the recombinant plasmids were extracted from positive colonies and sequenced. The results of nucleotide sequence indicated maximum of similarity with the same sequence in the Gallus gallus mitochondrial genome. Comparing these two sequences identified the presence of six nucleotide differences. However, only three point mutations led to the altering of dissimilar amino acids. Also, a protein BLAST query showed that the sequence had a similarity of 99% in 100% coverage to cytochrome C oxidase subunit 1. The protein BLAST query suggested the conserved domain cytochrome C, in a family which is part of a complex IV. This complex converts the oxygen into the water by electrons transfer from reduced cytochrome in a reaction associated with mitochondrial electron transport chain. The findings may be useful in identifying phenotypes in poultry.Keywords: Cloning, Sequencing, MT-COX1, Khorasan native poultry, Gallus gallu

Design and Production of a Novel Recombinant Chimeric IL2-Omp31 Antigen against Brucella Infection

Brucellosis is a zoonotic disease in human and animals. Brucella melitensis is one of the most pathogenic species of Brucella in goat and sheep. Omp31 is an outer membrane protein of Brucella that acts as an immunogenic protein. Cytokines are glycoproteins with low molecular weight that play the role of an immune adjuvant and regulate immune responses. Interleukin-2 is one of the most important cytokines, which are secreted by the white blood cells and involved in T cell immune responses. In the present study, a chimeric Omp31-Interleukin2 recombinant protein was generated by means of genetic engineering techniques. This chimeric coding sequence was amplified by using specific primers and using Splicing Overlap Extension (SOE) PCR technique. The fusion of the two mentioned proteins was accomplished using a rigid linker. The generated chimeric IL2-Omp31 fragment was TA cloned, and then subcloned into pEt22b vector as an expression vector. The chimeric protein was successfully expressed in E. coli BL21 (DE3) and confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and also Western-blotting analysis. Finally, in order to assess the antigenic features of the recombinant chimeric IL2-Opm31 protein, its secondary structure and antigenicity were predicted in silico

Assessment Association Between CAPN3 and ADRB3 Genes Polymorphism and Estimated Breeding Values (EBVs) of

The current study was designed to estimate the frequency of CAPN3 and ADRB3 genes single nucleotide polymorphisms (SNPs) and investigate if their polymorphisms have association with estimated breeding values (EBVs) of growth traits in Baluchi sheep. Phenotypic information about several sheep growth traits on 102 purebred Baluchi sheep was collected. Association effects of the variants of CAPN3 and ADRB3 genes on the growth traits were examined. Three conformational patterns were identified for CAPN3 but the ADRB3 gene was not polymorphic in our experimental population using PCR–SSCP analysis. Analysis of variance using the CAPN3 SNP as the independent variable and EBVs as dependent variable showed that the CAPN3 genotypes were associated with additive EBVs for weight at birth (P < 0.05) but no association of the CAPN3 genotypes with the other examined traits EBVs were found. Genotype BB had a superior birth weight when compared to those of individuals with other genotypes. Findings of this research suggest that polymorphisms in the CAPN3 gene might be one of the important genetic factors that influence growth traits and maybe explain partial source of genetic variation. The polymorphism may also be useful in marker-assisted selection for BW

Comparison of Fixed Regression and Random Regression Test-Day Models for genetic evaluation of milk yield trait in Holstein cows Razavi Khorasan province

The Fixed Regression Test-Day Model (FRM) and Random Regression Test-Day Model (RRM) for genetic evaluation of milk yield trait of dairy cattle in Khorasan Razavi province were studied. Breeding values and genetic parameters of milk yield trait from two models were compared. A total of 164391 monthly test day milk records (three times milking per day) obtained from 19217 Holstein cows distributed in 172 herds and calved from 1991 to 2008, were used to estimate genetic parameters and to predict breeding values. The contemporary group of herd- year- month of production was fitted as fixed effects in the models. Also linear and quadratic forms of age at calving and Holstein gene percentage were fitted as covariate. The random factors of the models were additive genetic and permanent environmental effects. In the random regression model, orthogonal legendre polynomial up to order 4(cubic) was implemented to take account of genetic and environmental aspects of milk production over the course of lactation. Heritability estimates resulted from the FRM was 0.15. The average heritability estimates resulted from the RRM of monthly test day milk production for the second half of the lactation was higher than that of the first half of lactation period. The highest and lowest heritability values were found for the first (0.102) and sixth (0.235) month of lactation. Breeding value of animals predicted from FRM and RRM were also compared. The results showed similar ranking of animals based on their breeding values from both models

Association of the polymorphism in the 5’ flanking region of the ovine IGF-I gene with growth traits in the Baluchi sheep

The insulin-like growth factor 1 (IGF-I) gene has been described in several studies as a candidate gene for growth traits in farm animals. The present preliminary study attempts to establish associations between growth traits and genetic polymorphisms at the 5’ flanking region s IGF-I in the Baluchi sheep. The DNA of 102 sheep of the indigenous Iranian Baluchi breed was evaluated. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of the 5’ flanking region (Exon1) of the ovine IGF-I gene revealed three banding patterns (genotypes) named as A/A, A/B and B/B. The evaluation of an association effect between these SSCP patterns with birth weight (BW), weaning weight (WW), and average daily gain from birth to weaning (GBW), weaning to six month (GWS) and from six month to yearling age (GSY) suggest a positive effect of pattern (Genotype) A/B with GBW and weaning weight (WW). Pattern (Genotype) B/B had a superior birth weight when compared to those of individuals with other patterns. Moreover, the A/A pattern (Genotype) appeared favourable for live weight at yearling age. These results confirmed the potential usefulness of this gene in marker-assisted selection programmes for sheep breeding

Cloning and molecular characterization of Omp31 gene from Brucella melitensis Rev 1 strain

Brucellosis, caused by the genus Brucella bacterium, is a well-known infection among domestic animals. Considering the serious economic and medical consequences of this infection, various preventive efforts have been made through using recombinant vaccines, based on outer membrane protein (OMP) antigens of Brucella species. The objective of the present study was to clone, analyze the sequence, and predict the epitopes of Omp31 gene as a major B. melitensis antigen. The full-length open reading frame (ORF) for this gene was amplified by specific primers and cloned into the pTZ57R/T vector. The gene sequence of B. melitensis Rev 1 strain was submitted to NCBI database. The results of phylogenetic analysis showed that Omp31 is almost similar in different Brucella species. Online prediction software programs were also used to predict B- and Tcell epitopes, secondary and tertiary structures, antigenicity, and enzymatic degradation sites. The bioinformatic tools in the current study were confirmed by the results of three different experimental epitope prediction studies. Bioinformatic analysis identified one T-cell and three B-cell epitopes for Omp31 antigen. Finally, based on the antigenicity and proteosome recognition sites, common B- and T-cell epitopes were predicted for Omp31 (amino acids 191-204). Bioinformatic analysis showed that these regions had proper epitope characterization and could be useful for recombinant vaccine development

Production and characterization of egg yolk antibody (IgY) against recombinant VP8 - S2 antigen

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. VP8 subunit of rotavirus is the major determinant of the viral infectivity and neutralization. Spike glycoprotein of coronavirus is responsible for induction of neutralizing antibody response. Studies showed that immunoglobulin of egg yolk (IgY) from immunized hens has been identified to be a convenient source for specific antibodies for using in immunotherapy and immunodiagnostic to limit the infections. In this study, chimeric VP8-S2 gene was designed using by computational techniques. The chimeric VP8-S2 gene was cloned and sub-cloned into pGH and pET32a (+) vectors. Then, recombinant pET32a-VP8-S2 vector was transferred into E. coli BL21 CodonPlus (DE3). The expressed protein was purified by Ni-NTA chromatography column. Hens were immunized with the purified VP8-S2 protein three times. IgY was purified from egg yolks using polyethylene glycol precipitation method. Activity and specificity of anti-VP8-S2 IgY were detected by dot-blotting, Western-blotting and indirect ELISA. We obtained anti-VP8-S2 IgY by immunizing hens with the recombinant VP8-S2 protein. The anti-VP8-S2 IgY was showed to bind specifically to the chimeric VP8-S2 protein by dot-blotting, Western-blotting analyses and indirect ELISA. The result of this study indicated that such construction can be useful to investigate as candidates for development of detection methods for simultaneous diagnosis of both infections. Specific IgY against the recombinant VP8-S2 could be recommended as a candidate for passive immunization against bovine rotavirus and bovine coronavirus