| A pair of RT-PCR primers were designed and synthesized based on the published gene sequence of chicken interleukin-2.The about 680bp target DNA sequence were amplified by polymerase chain reaction depending on the template of total RNA isolated from ConA-stimulated spleen cell,and cloned to pGEM-T vector.The gene fragment are identified to be interleukin-2 with the methods of restriction enzyme digestion,polymerase chain reaction,and sequencing. Comparing with the sequences of chickens reported in NCBI.there are mutation mucleotides of two within ORF.Hoever,amino acid analysis show that they share the same second-structure comparing with other interleukin-2 of other chicken species reported in GeneBank.The predicted second structure of encoding amino acid indicated,there is a leader sequence of 22 amino acid,and four conversed cysteines allowing the formation of intra chain dislfide,four α -helices being similarity to the ones of mammals. The phylogenetic analysis and IL-2 antigenic analysis indicated that chicken IL-2s have evolutionary relationship with mammals' IL-2s,so it confirmed the opinion that IL-2s have specificity of species.Using the technology of gene-engineering,the sequence coded ChIL-2 was subcloned to the expression plasmid pET28a vector, The recombined plasmid,it was named pET28a-GSIL-2,and confirmed by DNA sequence anylasis was transformed to BL21(DE3) and induced with IPTG..SDS-PAGE illuminated that the expressed protein was about 18 kD and reached its peak 5-7 hours after induction by IPTG at 37 ℃,which accoucted for 18% of total protein of host cell, .recombinant fusion protein was extracted roughly from dissolved BL21 and purified by affinity column. the biological activities of the rchIL-2 measured by concanavalin A (ConA)-induced lymphocyte proliferation assay,Using MTT The ability of rChIL-2 to cause both activation and proliferation of T cells in vivo indicates that it has the potential to be used as an immune activator and accine adjuvant.
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