Molecular Cloning, Expression And Bioactivity Verification Of Porcine Interleukin-34(pIL-34)

Interleukins are a group of proteins with important immune-regulatory functions that play an active role in improving immune state and resisting pathogen infection. Interleukin-34 (IL-34) is a novel interleukin firstly reported in 2008. By now, it has been known to be able to promote the viability of monocytes, to regulate the growth and differentiation of myeloid cells, and to facilitate the formation of osteoclast. Nevertheless, the knowledge on IL-34, including its physical and chemical characteristics, functions, working mechanism and expression regulation, is too limited, and there has not been any research reported about porcine IL-34. Consequently, this study, targeting on porcine IL-34, performed gene cloning and expression, and verified its bioactivity of promoting the proliferation of porcine peripheral blood mononuclear cells. The main contents include:1. The cloning and sequence analysis of porcine IL-34Based on a pig EST sequence sharing a high homology with human IL-34 mRNA sequence (CJ032290), a pair of primers pIL34-F/R was designed, and the cDNA of porcine IL-34 complete coding sequence was amplified via RT-PCR from total cellular RNA of PK-15 cells treated by poly(I:C). The sequence was cloned into pMD-18T vector and sequencing result showed that the cloned sequence was 708bp in length and encoded 235 amino acids. The sequence had a homology as high as 99.44% with the reference sequence in EST database. According to the sequence alignment result by Clustal W2, porcine IL-34 shared a homology of 78%,85%,71% and 71% with that of human, cow, rat and mouse, respectively, on the level of amino acid sequence.2. The tissue-specific expression analysis of porcine IL-34Using RNA of various tissues from a Meishan pig 6-week old as the template, semi-quantitative RT-PCR was performed to amplify IL-34, the target gene, and GAPDH, the internal reference gene, respectively, to analyse the expression difference of porcine IL-34 among different tissues. The result revealed that the expression level of porcine IL-34 mRNA differed between different tissues, and it was most abundant in small intestine, followed by brain. The expression could also be detected in spleen, lung and kidney, but was hardly found in stomach, liver and lymph.3. The prokaryotic expression of porcine IL-34 and the preparation of polyclonal antibodiesThe first 60 nucleotides of porcine IL-34 complete coding sequence encoding a signal peptide were removed via PCR, and the gene sequence for porcine IL-34 mature peptide was inserted into pET-28a(+) prokaryotic expression vector. The E.coli BL21 was transformed with the recombinant plasmid pET28-pI134, and the expression was induced by IPTG. The SDS-PAGE analysis showed that the fusion protein of porcine IL-34 with a 6×His tag was expressed in E.coli BL21, and its molecular weight was approximately 23kDa. The fusion protein was present in the form of insoluble inclusion body. The specificity of fusion protein was tested by Western blot, in which monoclonal antibody against 6×His tag was used as primary antibody. By using the expression product as antigen, New Zealand rabbits were immunized and rabbit polyclonal antibodies against porcine IL-34 were produced.4. The eukaryotic expression of porcine IL-34The eukaryotic expression plasmid pcDNA-pIL34 was constructed and transfected into CHO cells. Under the selection of G418, the CHO cell line stably expressing porcine IL-34 was constructed. RT-PCR and Western blot were performed, and the expression of porcine IL-34 was verified in the constructed cell line on mRNA level and protein level, respectively.5. The preliminary study of the bioactivity of porcine IL-34The culture supernatant of CHO cell line stably expressing porcine IL-34 and the culture supernatant of HeLa cell at different time points after transient transfection with pcDNA-pIL34 eukaryotic expression plasmid were collected. By MTT method, the bioactivity of porcine IL-34 to stimulate the proliferation of porcine peripheral mononuclear cells was verified. The result revealed that both of the two kinds of culture supernatants are capable of promoting the proliferation of PBMCs, but neither of them has a strong effect.