| PrefaceColorectal carcinoma is one of the common malignant tumors which has higherincidence, occupying the fourth place in our country and the death rate increasingcontinuously in recent years. Recent studies have shown that modulation of tumorantigen,expression shortfall of MHC antigen of the tumor cell and deficiency ofcytokine around tumor may be the main reasons which lead to immunodepression,which makes tumor escape from immune surveillance due to insufficient quantity andquality of lymphocyte. Therefore reversing the immunodepression and promoting theactivity of local immunocyte maybe a potential method to provocate strong anti-tumorimmune reaction.In the researches of looking for effective lymphocyte activators applied toanti-tumor, Super-antigen (SAg) is a neo-focus of anti tumor studies because of itscharacter to activate T cell dramatically. SAg, a kind of product of virus or bacteria, haspowerful ability to activate T cell without the presenting of antigen presenting cells. Itcan directly combine with both MHC-(?) and TCR v(?) region simultaneously and thenactivate CD4 T cell and CD8 T cell, which may directly kill MHC-(?) tumor cellconsequently, named SAg dependent cell-mediated cytotoxicity (SDCC). The activatedT cells can release lots of cytokine to kill tumor cells or stimulate them to expressMHC molecule which can cause NK cell to kill tumor cells. Staphylococcus enterotoxin A (SEA), an enterotoxin produced from staphylococcus, is a kind of SAgresearched extensively among SAg family. Many experiments in vivo and vitro haveidentified that SEA has obvious function to provocate antineoplastic immune response.Although SEA has a strong ability to activate T cell and inhibit tumor cellsobviously, SEA is lack of antigenic specificity, killing MHC(?) normal cells besides allof tumor cells can express MHC (?) molecule, which limited its application in clinicaltreatment. Recently fusion protein of SEA and MAb constructed by gene engineeringhas both strong immune activation from SEA and targeting function from MAb tocorresponding antigen expressed on tumor cells without the help of MHC (?) molecule,so it can activate lots of T cells designedly to release many kinds of cytokines in tumormicroenvironment, named MAb targeted SEA. Recently, a kind of small fragmentmolecule such as ScFv and Fab' has been reconstructed from the complete MAb as theantibody fraction, overcoming the defects of complete MAb's defects as high moleculeweight,low penetration and high immunogenicity, and only remaining similarantigenic specificity as well as easy to be constructed on gene level, it has been appliedto construct fusion protein of SEA and ScFv broadly and shown remarkablecytotoxicity.ND-1 MAb is a murine monoclonal antibody against tumor-associated antigenLEA, mainly expressed in human colorectal carcinoma, obtained by immunized Balb/cmice with CCL-187 human colorectal carcinoma cell line. ND-1 can bind specially towell differentiated and moderately differentiated colorectal carcinoma tissues. Singlechain Fv ND-1 ScFv, constructed by fusing gene of variable fragment of heavy chainand light chain, can express in the surface of E.CoLi cells and has similar affinity toCCL-187．Therefore, in this study, the fusion gene of ND-1ScFv and SEA has beenconstructed, expressed in E.coli cells and exhibiting cytotoxicity successively. Theconstruction of fusion protein provides an evidence for both nude mice bearingexperiments and clinical immunotherapy of colorectal carcinoma. Materials and MethodsFrom the staphylococcus aureus, the genome DNA of SEA was extracted and usedfor PCR to amplify the SEA gene by primer P1 and P2．Then PCR product was ligatedinto clone vector of pMD18-T. After DNA sequencing, from the pMD18-T SEA vector,the gene of linker-SEA was amplified and added a linker sequence(encoding GS) tothe upstream of the gene SEA and restrict sites Sal (?) and Hind (?) by primer P3 and P4 .PCR product was ligated into clone vector of pMD18-T to form pMD18-T linker-SEA.Positive clone containing pMD18-T linker-SEA was identified by DNA sequencing.Then, from the vector pET-28a(+)ND-lScFv, the gene of ND-1ScFv wasamplified and add restrict sites BamH (?) and Sal(?) by primer P5 and P6．PCR productwas ligated into clone vector of pMD18-T to form pMD18-T ND-1ScFv . Positiveclone containing pMD18-T ScFv was identified by DNA sequencing. pMD18-TND-1ScFv was double digested by BamH (?) and Sal(?) ,the product ND-1ScFv isrecovered and linked into pQE30 vector which is prepared by the same doubledigestion to get the expressing vector pQE30 ND-1ScFv.pMD18-T linker-SEA was double digested by Sal(?) and Hind (?) and the productlinker-SEA was rocovered and ligated into pQE30 ND-1ScFv which is prepared by thesame double digestion. the novel expressing vector pQE30 ND-1ScFv-SEA wastransformed into E.coli M15．In the condition of IPTG(final concentration1mM),25(?),induce 3 hours the ND-1ScFv-SEA was expressed. Target protein waspurified by Ni-NTA resin affinity chromatography and then refold it. Its molecularweight and purity were analyzed by SDS-PAGE. Immunofluorescence Assay wasconducted to determinate the specificity of ND-1ScFv-SEA to CCL-187 by comparingto HeLa cell line. The cytoxicity of ND-1ScFv-SEA to CCL-187 compared to SEA wasdetected by MTT.ResultsUsing the genome of Staphylococcus aureus as template, PCR got the gene ofSEA , the production is about 770bp which was compatible to designed sequence and proofed by the DNA sequencing. Using the vector pMD18-T SEA as template, PCRgot the gene of linker-SEA and added BamH (?)/ Sal I. The production is about 730bpwhich was compatible to designed sequence proofed by the DNA sequencing. Theplasmid pMD18-T linker-SEA is digested by BamH (?)/ Sal I and also got a 730bpfragment.Using the vector pET-28a(+)ND-1ScFv as template, PCR got ScFv and addedSal I/Hind (?). The production is about 750bp ,which was compatible to designedsequence proofed by the DNA sequencing . The plasmid pQE30 ND-1ScFv isidentified by double digestion and also got a 750bp fragment.pMD18-T linker-SEA is digested by Sal (?) /Hind (?) and rocovered the productof linker-SEA and ligated it to pQE30 ScFv to form the expressing plasmid pQE30ND-1ScFv-SEA. Identify the plasmid by BamH (?) /Hind (?) double digestion and got a1500bp fragment, which show the expression plasmid is constructed correctly.After transform the expression plasmid into E.coli M15, the fusion protein wasinduced to exression by 1mM IPTG at 25(?) for 3 hours. Harvest inclusion body, thenpurified by Ni-NTA resin affinity chromatography. SDS-PAGE showed that the purityof purified ND-1ScFv-SEA is more than 90% and molecular weight is near 55kDawhich is compatible to its theoretic weight. Immunofiuorescence Assay showed thatpurified ND-1ScFv-SEA had strong specificity to CCL-187 cell line. MTTdemonstrated that the cytotoxicity of ND-1ScFv-SEA is remarkable, about 91%,which is much higher than SEA.ConclusionWe have successfully constructed the fusion protein ND-lScFv-SEA ofsuperantigen SEA and single chain Fv of ND-1ScFv. Results indicated that thecytotoxicity of ND-1ScFv-SEA to CCL-187 is remarkable and it may be a good reagentfor colorectal carcinoma immunotherapy form. |
