Gene Cloning,Expression And Functional Analysis Of Of Human Chemokine MIP-3 Alpha

Macrophage inflammatory protein 3-α(MIP-3α) is a member of chemokine superfamily.it is mostly secreted by epidermis cell or cell,The main function of MIP-3αis attract Langerhans Cells(LC) to epidermis. As well know , LC is mostly important antibody precent cell, and it decide character of immunity reaction in the tissue of skin, it is also main reason of immunity exclude reaction after transplant variant skin. Simultaneity , MIP-3αis attract immature DC and lymphocyte cell,thereby ,MIP-3αis important actor in imflammation and tumour,as one of important part of chemokine,more and more attention was attracted by people.but it is first step to study of MIP-3α. Thereforewe cloned the human MIP-3αmature protein gene and constructed it in a prokaryotic expression vector pET32a(+).The E.coli. cells transformed with pET32a(+)/MIP-3αwere cultured and induced with IPTG to express the corresponding fusion protein TrxA- MIP-3α.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 tumour therapy,transplant exclude reaction, immunity and so on . On the other hand ,wo can find someones mutation protein which can resist CCR6, than observe the function of it on release inflammation occur or reduce of exclude from skin.The results are as follows:1. The total RNA was isolated from the human inflammatory tonsil and the cDNA encoding the mature protein of MIP-3αwas 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 MIP-3αnature 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 25kDa recombinant fusion protein was...