| Scavenger receptors, as one family of innate immune receptors, play an important role in host defense against microbial infections. However, to date, little is known about porcine scavenger receptors in immune host defense against microbial infections.Objective:To gain insights into the biological functions of porcine scavenger receptor SRA and MARCO and reveal the role of them in immunity,we took scavenger receptor A SRA and MARCO of pig as the research objects, using prokaryotic expression system to express the extracellular domain, and to produce the recombinant protein which has been purified. We took the recombinant protein as immunogen to make polyclonal antibodies which could be used to verify the specificity of the antiserum. In addition, we isolated some bacterial pathogens from porcine lungs, and then took advantage of stable CHO cell lines, named CHO-SRA and CHO-MARCO, to reveal its biological function.Materials and Methods: ①Firstly a CDS gene was amplified by RT-PCR from porcine lungs and cloned into p MD18-T vector, named p MD18-SRAwt and p MD18-MARCOwt,respectively. Subsequently, a gene fragment, which encodes extracellular domain of porcine SRA or MARCO, was amplified by PCR from p MD18-SRAwt or p MD18-MARCOwt, and cloned into p ET-28 a to construct the recombinant plasmid, named p ET-r SRA-c or p ET-r MARCO-c. The recombinant protein, named r SRA-c(about 32 k Da) and r MARCO-c(about 31 k Da) respectively, mainly expressed as inclusion body, was purified by His·Bind®affinity chromatography. Furthermore, BALB/c mice were immunized by purified r SRA-c or r MARCO-c for generation of polyclonal antibodies. ② To determine the role of porcine SRA/and MARCO in pulmonary disease, the CDS was cloned into PCDNA3.0 vector to construct the eukaryotic plasmid. Subsequently the recombinant plasmid was transfected into CHO cells passed on 96 wells and filtered by G418 to construct stable CHO cell lines, named CHO-SRA and CHO-MARCO, and was verified through RT-PCR and Western blot. ③ To dissect clinical onset pigs and isolate bacteria from the lungs, interacting bacteria marked by FITC with the stable cell lines, and thus to analyze the function of SRA and MARCO.Results: ① The result of amplification showed that SRA’s fragment size was 1341 bp, and MARCO’s fragment size was 1188 bp, which was consistent with the expected results. The sequencing results are as same as the original coding sequence, indicating that the amplified sequence was correct. Prokaryotic plasmid PCR validation showed that SRA and MARCO fragment sizes were 711 bp, 709 bp, and the same as the fragment size. The sequencing results were in line with the sequence of the extracellular domain, showing that the prokaryotic expression plasmid was successfully constructed. Western blot analysis indicated that the size of SRA and MARCO appeared to be 32 k D and 31 k D respectively, which matched the reference. Meanwhile the results of serology showed that antiserum can identify r SRA-c(about 32 k Da) and r MARCO-c(about31k Da), having a good immunogenicity. ② In the results of eukaryotic plasmid PCR validation, SRA and MARCO sizes were 1341 bp and1188bp, respectively. Consistent with the size of the coding sequence, the sequencing results showed that eukaryotic expression plasmid had been successfully constructed. RT-PCR showed that SRA cell lines the band size was 1341 bp, which matched the CDS length of SRA.In the MARCO cell lines, the bands size was 1188 bp, which was the same of CDS size of MARCO. Western blot analysis of cell lines displayed that SRA+ clone cell contained protein with size of 55 k D, and MARCO+ clone cell contained protein with size less than 55 k D, which showed that stable cell lines had been constructed successfully.③We used stable CHO cell line, named CHO-SRA and CHO-MARCO respectively, to identify the interaction between porcine CHO-SRA or CHO-MARCO and bacterial pathogens which were isolated from lungs of pigs with pulmonary symptoms. Our results showed that porcine SRA/CD204 and MARCO both mediated phagocytosis of bacteria like E.coli, S. aureus and S. pyogenes, but not Actinobacillus pleuropneumoniae(APP).Collectively, our results could provide a basis for identification of effects of pig SRA and MARCO on microbial infections and for further study of the role of porcine SRA and MARCO in pulmonary disease. |
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