| The immunotoxin is a special anti-minor medicine,it can exactly combine to the special-expressed antigen or acceptor on the tumor cell surface guided by the anti-body or cell-factor,and then destroy the tumor cell.Melittin,as a typical cationic antimicrobial peptides(CAP),is physiologically active.This project aimed at expressing melittin in E.coIi,by constructing a membrane-toxic immunotoxin, RGD-Mel,in which melittin was toxic part and RGD(Arg-Gly-Asp)was target part. The fusion protein was expressed using pET30a vector,and its activity was tested in vitro.The main results were as followings:The total RNA was extracted from venom gland of Apis cerana cerana worker at melittin highly expressed period,and the mature fragment of Melittin cDNA were cloned by RT-PCR using Taq DNA polymerase.We discovered that a more complete and correct cDNA could be cloned at higher reverse transcriptase temperature using AMV compared to at 37-42℃.The mature fragment of Melittin cDNA from A.c.cerana were cloned by using pUCT-vector developed from pUC18-vector.The cloned sequence has only one base difference at 33rdbase from that of A.m.ligustica,though the base difference had no influence to the sequence of amino acid.It meant that the Melittin amino acid sequence of A.c.cerana was completely identical to that of A.m.ligustica.The fusion protein of RGD-Melittin was effectively expressed in E.coli using PET30a,and purified.It can kill or wound tumor cells,When the concentration of melittin was 5.0μg/mL,the suppression rate of tumor cells was 39.6%.However, the suppression rate was 45.5%,when was testd at the same concentration.The suppression rate of RGD-Mel at 3.0μg/mL was the same as that of nature melittin at 5.0μg/mL.RGD-Mel is an anti-tumor immunotoxin targeting tumor cell membrane in vitro, however its function in vivo still need to be tested.This research have the latent application value in the tumor guidance treatment and the clinical aspect.
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