Gene Cloning, Expression Of The Human Chemokine SLC And Its Functional Analysis

Secondary Lymphoid-Tissue Chemokine(SLC) is a member of chemokine superfamily. SLC can attract T cells and DCs into the secondary lymphoid tissues ,where DCs present antigen to T cells and activate T cells.So SLC is an important chemokine to elicite the host specific immunity. Studies have revealed that lack of mouse-SLC in mouse can lead to the mistake of localization of DCs and homing of T cells,as well as delayed activation of T cells and disturbance of the structure of secondary lymphoid tissues.However,overexpression of SLC always results in progression of many kinds of autoimmue disorders.Therefore,we may developed a new strategies based on its bio-activities to treat transplant rejection and relevant diseases.For this reason ,we cloned the human SLC mature protein gene and constructed it in a prokaryotic expression vector pET32a(+).The E.coli. cells transformed with pET32a(+)/SLC were cultured and induced with IPTG to express the corresponding fusion protein TrxA-SLC.Then, the protein was purified with TALON resin and weak cation exchange chromatography,and its biological functions were assayed.This research makes further studies on novel therapeutic strategies possible. The results are as follows:1. The total RNA was isolated from the human inflammatory tonsil and the cDNA encoding the mature protein of SLC was amplified with RT-PCR and the DNA fragment was inserted into pET32a(+).The 3 amino acids between target gene and the Enterkinase site were deleted by mutation and the recombinant vector of SLC nature protein was constructed and the sequence was verified by DNA sequencing. The sequence was consistent with that of the report in GenBank.2. The recombinant vector was transformed into E.coli. BL21 trxB(DE3).The expression was induced by IPTG and optimal conditions of induction were achieved.SDS-PAGE analysis showed that the about 30kDa recombinant fusion protein was expressed and the proportion of the fusion protein was about half of the total protein of induced bacteria.And about 50% of the fusion protein existance in the supernatant of the lysate.The expressed protein was identified by Western Blotting.3. The expressed protein was enriched with TALON resin and the SLC was released with enterokinase cleavage,purified with weak cation exchange chromatography. SDS- PAGE analysis showed that the target protein was highly purified.4. The biological activity of the purified,recombinant protein was assayed with chemotaxis test and calcium flux test.The purified protein could strongly chemoattacted the human peripheral blood CCR7-bearing lymphocytes and elicite the calcium fluxing of the cells.The functional recombinant human SLC protein was successfully produced, paving the way for further study on mechanism of SLC and novel therapeutic strategies based on SLC.