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Application of Long-term cultured Interferon-γ Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle
Published on: July 11, 2015
Interferon stimulated gene 15 (ISG15): molecular characterization and expression profile in endometrium of buffalo
Asit Jain1, Pradyumna S Baviskar, Sukumar Kandasamy
1Genome Analysis Laboratory, Animal Genetics Division, Indian Veterinary Research Institute, Izatnagar, India.
Abstract:
Interferon stimulated gene 15 (ISG15), one of the several proteins induced by conceptus derived Type I and/or a Type II interferon (IFN), is implicated as an important factor in determining the uterine receptivity and conceptus development. However, presence as well as specific role of the ISG15 in buffalo (Bubalus bubalis) reproduction is yet to be elucidated. In the present study, both genomic and cDNA sequences of bubaline (bu) ISG15 were cloned and investigated for its expression in different tissues of female reproductive tract of buffalo. Sequence analysis revealed 100% identity among the genomic sequences (1014 bp) of buISG15 from three different breeds of buffalo (viz., Murrah: Acc. No. DQ118137, Mehsana: Acc. No. DQ118138, and Nagpuri: Acc. No. DQ118136) and cDNAs (Acc. Nos. HM543268-HM543270). As in cattle, the buISG15 was comprised of two exons of 57 bp and 520 bp encoding a peptide of 154 amino acids. Moreover, the buISG15 cDNA sequence exhibited 98.3% and 98.5% identity with that of taurine and indicine cattle, respectively. Subsequent reverse transcription PCR analysis revealed expression of the buISG15 in the uterine endometrium, corpus luteum (CL), corpus hemorrhagicum and oviduct. Quantitative Real Time PCR (RTqPCR) analysis also confirmed the constitutive expression of the buISG15 in the uterine endometrium during different stages (i.e. estrus, diestrus and proestrus) of estrous cycle and also during early (∼d 30-40) pregnancy. Western blot analysis of the endometrial extract from both estrous cyclic as well as pregnant buffalo demonstrated the presence of only conjugated ISG15 which was >40 kDa. ISG15 mRNA and immune-reactive proteins were localized in the stromal as well as glandular epithelial cells of the uterine endometrium of estrous cyclic as well as pregnant buffalo. However, there was no significant difference in amount of ISG15 mRNA across the different reproductive phases. To conclude, this study will be helpful for the further understanding of the roles of the ISG15 in pregnancy of buffalo cows.
