The Expression Of SIL-16 In E.coli And Its Activity Analysis

Interleukin-16(IL-16) is a pleiotropic proinflammatory cytokine originally identified by Center and Cruik　Shank in 1982. Human IL-16 is predominantly synthesized by CD8+ T lymphocytes as a 631 amino acid precursor that is enzymatically cleaved between Asp 510 and Ser 511 to yield the c-terminal 121 amino acid bioactive molecule, that is sIL-16 (secreted IL-16).sIL-16 forms a PDZ domain which is classically associated with intracellular protein that bind the c-terminal peptide motifs of membrane or structural proteins. sIL-16 might be the first example of a secreted PDZ protein. It does not contain a secretory leader sequence, which is similar to interleukin-1(IL-1), although there is no homology between the two cytokines. The NMR structure of recombinant IL-16 predicts a monomeric form while full bioactivity is associated with tetramer formation.sIL-16 is a chemoattractant for CD4+ T cells, and the effect is observed equally in activated and resting T cells. sIL-16 induces a G0 to G1 cell cycle change associated with the expression of IL-2R on 20 to 100 percent of human blood CD4+T cells, which are then competent to proliferate in the presence of exogenously added IL-2 and IL-15. It has been reported that sIL-16 inhabits the transcription of the HIV long terminal repeat through CD4- dependent signals that rely on the presence of the HIV core enhancer and the drop in sIL-16 level has an intimate relationship with AIDS progression.The recent report suggested sIL-16 takes part in the regulation of asthma at the early stage. sIL-16 production may be maintained by an antocrine machinery by epithelical cell-derived IL-16, thus accelerates air tract inflammation.Methods and results: At first the sIL-16 cDNA was amplified by PCR, and then it was digested by NdeI and XhoI. the fragment was cloned into expression vector pET28a(+) which was also digested with NdeI and XhoI. the resultant plasmid not only included 6×His at the N-terminal, but also was under the T7 promoter control. The plasmid was transformed into BL21(DE3) for the expression of rIL-16. E.coli cells were harvested after induction by IPTG and there was higher productivity at 20℃ than at 37℃. After inducing for 12 hours at 20℃,the bacterium were sonicated. The supernatant was analysed by SDS-PAGE after the centrifuging. An obvious expression about 18kD can be detected. It also be shown by western blotting which use IL-16 polyclone antibody as the first antibody. Then the supernatant was purified by Ni iron affinity chromatography column according to imidazole level change and the purity up to 90 percent. After the recombinant protein frozen and dried, the activity was detected by stimulating PBMC surface IL-2R expression. And the experiment suggested that the unstimulated cells were 11.46 percent IL-2R and this increased to 30 percent following 10-9M rIL-16 stimulation.Conclusion: construct the high effect expression vector pET-sIL16, the recombinant protein was gained after IPTG inducing and its productivity up to 40 percent of the total products; the protein purity was finished by Ni iron affinity chromatography column because the vector 6×His at the N-terminal, and the effect was up to 90 percent. The low temperature induction lessen protein molecular hydrophilic reaction, which favored the protein fold and nature formation. It proved the rIL-16 activity since PBMC surface IL-2R expression increased 18.54 percent after rIL-16 stimulation.