Cloning And Expression Of Three Swine Cytokines And Immunologic Enhancement Of Three Goat Cytokines

Cytokines are a kind of regulation factors which have broad bio-activities and can activate the immune cells and involve in immune response. They play a significant role in the generation and regulation of immune response in vivo. Interleukin-2(IL-2)? Interleukin-4(IL-4)and Interferon-?(IFN-?) are important to the development of humoral immunity responses, cell-mediated immune responses, defending the infection and resisting the tumor.This paper there cytokines of goat and porcine, and studied the biological activity of cytokines.We constructed the prokaryotic expression vectors and eukaryotic expression vectors of cytokines.Then detected the RT-PCR and westen-blot in mice, and studied the immunologic enhancement about FMD of goat's cytokines.This research is a good foundation for future research and application.1.Cloning IL-4 and IFN-?of swine In this research, two pairs of specific primers were respectively designed according to the sequences of swine IL-4 and IFN-? published in Gen Bank,target genes were amplified by using RT-PCR from swine PBMC stimulated by Con A.Sequence analysis of mature IL-4 showed that it contained 321bp and encoded 107 amino acids. The sequencing of mature IFN-?genes showed that IFN-?was 432bp and encoded 144 amino acids.2.Expression of the recombinant swine's cytokines There cytokines of swine genes were inserted into expression vector of pET32a(+) or pET28a(+),and obtained the recombinant protein.Recombinant swine IL-2 was about 21.4 KD,recombinant swine IL-4 was about 30.3 KD and recombinant swine IFN-? was about 18.4 KD.The recombinant protein were renatured with dialysis after purified by Ni-Ni metal chelate affinity chromatography. MTT was used to examine the biological activity of the recombinant protein.. The results indicated that the purified recombinant protein has good bioactivity which induced the proliferation of activated T lymphocytes in porcine's PBMC,and the typically demonstrated 4 ug/ml?8 ug/ml of activity. The activity of IFN-? in PK-15/VSV is 2.70×103U/mg.3.Constructing eukaryotic expression vectors Genes were inserted into eukaryotic vector of pVAX1.0,got the recombinant expression plasmid.The recombinant plasmid were immuned mice by intramuscular method and detected the target genes and proteins by RT-PCR. and westen-blot.4. Studied immunologic enhancement of goat cytokines Immuned goats with three recombinant cytokines and FMD vaccine,after immunization we detected antibody levels and IL-2?IL-4?IL-10?IL-17?EFN-? after immuned.The result shows that the recombinant protein can increase the levels of antibody and cytokines.