Molecular characterization, tissue expression, and antiviral activities of Bama minipig interferon-α subtypes

Abstract

Interferons play a major role in innate immunity and disease resistance. Porcine interferon alpha has 17 subtypes, and their gene sequences, tissue expression profiles, and antiviral activities have been primarily studied in domestic pigs but not in minipigs. Bama minipigs are genetically stable disease-resistant and making them as laboratory animal models for bioscience studies. To define the potential mechanism for disease resistance, in this study, we cloned 17 subtypes of Porcine interferon alpha genes in Bama minipigs using high fidelity polymerase chain reaction and subsequent sequencing. Sequence alignment showed that the 17 porcine interferon alpha subtypes were 98%-100 % homologous in those of domestic pigs. However, significantly different tissue expression profiles of PoIFN-α subtypes were found in the two pig species using real-time quantitative RT-PCR. Among the 10 different Bama minipig tissues tested, significant expression of multi-subtype porcine interferon alpha was detected in the lymph nodes and spleen, whereas no or low expression of fewer subtypes was detected in the heart, lung, brain, and small intestine. Sequence analysis revealed that the porcine interferon alpha promoters were almost similar between the two pig species. A cytopathic effect inhibition assay showed that the recombinant 17 porcine interferon alpha subtypes purified from mammalian cells had significantly different antiviral profile against vesicular stomatitis virus, porcine pseudorabies virus and porcine reproductive and respiratory syndrome virus compared with those in domestic pigs. Our findings provide evidence that porcine interferon alpha subtypes are highly conserved between Bama minipigs and domestic pigs but show varied tissue expression pattern and antiviral capabilities, which may contribute to their differences in disease resistance.

Keywords: Antiviral activity; Chinese Bama minipigs; Gene cloning; Porcine interferon-α subtypes; Tissue expression analysis.

Fig. 1

Sequence alignment of Bama minipig PoIFN-α promoters. The promoter regions were amplified from gDNA using sequence-specific primer pairs. The four promoter modules and TATA box are boxed. Short lines indicate nucleotide deletions.

Fig. 2

SDS-PAGE and western blotting analyses of the recombinant proteins of the 17 Bama minipig PoIFN-α subtypes. The recombinant proteins were expressed as His-tagged proteins and purified using Ni-NTA affinity chromatography. The purity was assessed using SDS-PAGE.

Fig. 3

Comparison of antiviral activities of the 17 Bama minipig PoIFN-α subtypes against three different pig viruses at two different doses. The antiviral activities were detected by CPE inhibition assay using the purified recombinant proteins at a dose of 2 ng/ml (A) or 200 ng/ml (B). Different letters indicate significant differences compared with virus control (VC).