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Cloning, Expression and Bioactivity Analysis of Goat Interleukin-1β and Interleukin-6

Author: LiXueZuo
Tutor: LiXiangRui
School: Nanjing Agricultural College
Course: Preventive Veterinary Medicine
Keywords: goat IL-1β IL-6 bioactivity
CLC: S827
Type: Master's thesis
Year: 2010
Downloads: 10
Quote: 0
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Abstract


Interleukin-1β(IL-1β) is one of the most important mediators that induce a casca-de of leading to inflammation. In mammals, IL-1βis mainly produced by monocytes and macrophages, and enhances cell-mediated immunity by inducing the growth and proliferation of lymphocytes. IL-1βalso has the potential to enhance the immune res-ponse induced by a vaccine. So recombinant IL-1β(rIL-1β) is currently being studied or used as an adjuvant for vaccines in sheep, pigs, cattle, and fish.Interleukin-6 (IL-6) is a pleiotropic cytokine that plays major roles in regulating immune response, acute phase reactions, haematopoiesis, and inflammation. It is pro-duced by many different cell types and acts on B cells, T cells, hepatocytes, haemato-poietic progenitor cells, and cells of the central nervous system. IL-6 is a potent indu-cer of the acute phase response, along with IL-1 and TNF-a.In this research, two pairs of specific primers were respectively designed accordi-ng to the sequences of goat IL-1βand IL-6 published in GeneBank, and goat IL-1βa-nd IL-6 were respectively amplified. Sequence analysis of GIL-1βdemonstrated an o-pen reading frame (ORF) of 801 bp in length and encoded 267 amino acids, which sh-ared nucleotide sequence similarity of 99.4% with that of GIL-1βin GenBank. The O-RF of precursor goat IL-6 was 627bp and mature goat IL-6 was 552bp in length and encoded 184 amino acids, which shared nucleotide sequence similarity of 99.6% with that of GIL-6 in GenBank.By the means of DNA recombination technique, GIL-1βgene was cloned into pr-okaryotic expression vector pET28a (+) and transformed into BL21 (DE3), and mGIL-6 gene was cloned into prokaryotic expression vector pET32a (+) and transformed i-nto BL21 (DE3). The recombinant protein was expressed by inducing with IPTG. As the results of SDS-PAGE indicated, the recombinant protein of GIL-1βand mGIL-6 were successfully expressed in E. coli with respectively the relative molecular weight of about 33.07 KDa and 42.24 KDa. The results of SDS-PAGE of the pellet and supe-rnatant after ultrasonication showed that most of the recombinant protein of GIL-1βe-xisted in the inclusion body and that most of the recombinant protein of mGIL-6 exis- ted both in the supernatant and inclusion body.In order to examine the biological activity of the recombinant protein of GIL-1β, The effects of Co-concanavalin A (ConA) with rGIL-1βon proliferation of C57BL rat thymocyte was determined. The results indicated that the recombinant protein of GIL-1βhas good bioactivity. To explore the biological activities of the recombinan protein of mGIL-6, Concanavalin A (ConA) induced and rmGIL-6 stimulated goat peripheral blood mononuclear cells (PBMC) proliferation assay was carried out. The results indicated that the recombinant protein of mGIL-6 significantly stimulated ConA-induced PBMC proliferation.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Livestock > Goat
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