Preliminary identification of the cloning,expression,and function of Marmota himalayana type Ⅰ interferon receptor β subunit
Objective To clone the gene of Marmota himalayana type Ⅰ interferon receptor β subunit(mhIFNAR2),and to perform antibody preparation and functional identification.Methods RT-PCR was used for amplification in the spleen tissue of Marmota himalayana to obtain the sequence,which was cloned to the prokaryotic expression vector pRSET-B to express the recombinant protein.Electrophoresis and Western blot were used for identification.BALB/c mice were immunized with the recombinant protein to prepare the polyclonal antibody of its extracellular domain;immunohistochemistry,immunofluorescence assay,and Western Blot were used for identification,and the method of siRNA blockade was used to investigate its function.An analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference t-test was used for comparison between two groups.Results A fragment of mhIFNAR2(149—1 300 bp)was obtained from spleen tissue,which showed the highest homology of 98.05%in marmot.A prokaryotic expression plasmid was successfully constructed for expression of the extracellular domain of the mhIFNAR2(50-181aa)and was named pRSET-B.mhIFNAR2,and the recombinant protein expressed by this plasmid had a molecular weight of 27 kD,a purity of about 95%after purification,and a concentration of 160 μg/mL.After BALB/c mice were immunized with the purified recombinant protein,1∶1 000 specific polyclonal antibodies were obtained,and immunohistochemistry and immunofluorescence assay showed the expression in cell membrane and cytoplasm.Among the three siRNAs synthesized,the siRNA starting from the 277 locus(siRNA277)could silence the expression of target genes and weaken the interferon signaling pathway compared with the blank control group and the negative control group(both P<0.05).Conclusion The fragment of mhIFNAR2 is obtained,and the polyclonal antibody for the extracellular domain of mhIFNAR2 is successfully prepared,with relatively high titer and specificity,and can be used for immunohistochemistry,immunofluorescence assay,and Western blot.
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