| Up to now, there are still some infectious diseases that cannot be prevented effectively. The traditional vaccines are mostly less safe or efficacious, driving efforts to develop vaccines of new generation. The previous studies have shown that immune response to an antigen will be enhanced greatly when some cytokines are pre-injected or injected together with the antigen. In order to get better understanding of the pathogensis of infectious diseases, it is valuable to study the relationship between cytokine profiles and the outcome of the diseases. The secreted cytokines play an important role in activating a variety of lymphoid cells, including B cells, cytotoxic T cells, natural killer cells, macrophages and additional cells, which participate in the immune response. Based on the cytokines produced by lymphoid cells, two cell populations have been further identified in CD4+T cells. Th1 cells produce interleukin (IL)-2, interferon (IFN)-γ and tumor necrosis factor (TNF)-β, while Th2 cells secrete IL-4, IL-5, IL-6 and IL-10. Different cytokines direct the polarization of Th cells and are instrumental in pivoting the development of an immune response. Porcine IL-10 (pIL-10), also called cytokine synthesis inhibitory factor (CSIF) and B cell-derived T cell growth factor (B-TCGF), is secreted by Th2 cells, and can inhibit the expression of Th1 cytokines. Porcine IL-12 (pIL-12), also termed as natural killer cell stimulatory factor (NKSF) and cytotoxic lymphocyte maturation factor (CLMF), can induce some cytokines, particularly IFN-γ, from T and NK cells, as well as enhance the cytotoxic activity of these cells. Its most critical role may be to initiate the development of cell-mediated immunity by promoting the differentiation of Th1 cells from naive T cells. IL-12 is an effective adjuvant that can initiate protective immune response. It regulates specific and nonspecific immune response by activating natural killer cells and cytotoxic T lymphocytes. In recent decades, extensive researches have been done and good progress have been made. Many cytokine genes have been cloned and expressed. The genes of porcine IL-10 and IL-12 have been cloned and expressed in prokaryotic expression system and in eukaryotic expression system abroad, but no report is available in China. Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) stimulated by LPS. Then the cDNAs of IL-10 gene and the IL-12 genes of two subunits were amplified by reverse transcription-polymerase chain reaction (RT-PCR) using specific primers, and then cloned into the vectors. The sequence analysis showed that the sequence of IL-10 is identical to that published in GenBank. Its open reading frame is composed of 528 base pairs (bp) and predicted to code for a peptide of 175 amino acids. The gene was subcloned into the pPROEXTM HTb vector, then was transformed the vector into E.coli DH5α. A biologically active recombinant pIL-10 was obtained after induction by IPTG. The cDNAs of the two subunits of IL-12 consist of 720bp(p35)and 1044bp(p40)and encoding peptides of 223 and 325 amino acids, respectively. The sequence of p35 of pIL-12 was the same as that of the published in GenBank. But the gene of p40 had 4 nucleotide differences from the published one, suggesting that the p40 of pIL-12 may be polymorphic. The two genes were subcloned into pET30 vector, and recombinant proteins with predicted molecular weight were achieved. In this paper, we constructed the recombinant E.coli highly expressing pIL-10 in order to study the pleiotropic immunological regulations in various diseases. The recombinant pIL-10 can be a valuable therapeutic agent for the diseases with overproduction of inflammatory cytokines. We also constructed the recombinant E.coli strains that highly express pIL-12 two subunits with immunological activity. They can be used to generate monoclonal antibody against pIL-12.
|
PDF Full Text Request