Construction Of Expressing Vector Of GPI-CD80, Expression In COS-7 And Preliminary Study Of Its Anchoring Ability

BackgroundTumor is one of the malignancy diseases threatening the health of human beingseriously. Following with the development of science and technologies, therealization of researchers for tumor's pathogenesies and therapia strategies had beenincreasingly deepened, from tissue and organ levels to protein and nucleic acid levels.In 1950s, researchers discovered that tumor associated antigens (TAA) existed inspontaneous and introduced animal tumors, and TAA could introduce specificimmune response generated by bearing cancer individuality. According to this,Burnet and Thomas proposed immunosurveillance theory in 1950 and 1960. Themain point of this theory is that, immune system may monitor the development oftumor cells and kill them by cell immunologic mechanism, so whenever the immunefunction is descendent, tumor would develop. The mechanisms of tumor cellsescaping immune surveillance of the body include that, (1) mutation of tumorantigens; (2) down-regulated surface MHC molecules; (3) defective antigenpresentation process; (4) lack of cell factors or externalization of immune suppressionfactors; and (5) others: Many tumor cells synthesize a multitude of glycocalyix which is similar to cell wall of bacterium. At present, gene therapy provides us a newtherapeutic tool to treat some refractoriness diseases. Gene therapy to tumor is mainlyby introducing some gene with therapeutic value, which will lead to generatingeffective immune response to resist tumor. These exogenous genes include thatcoding genes of some immune factors (such as cytokines, MHC and costimulatoryfactors), antioncogene, antisense oligo-nucleotides, tumor medicine related gene andvirogene, et al.Anti-tumor immune mainly depend on the lethal effect exercised by CTL. Inorder to generate potent cell-mediated immune response, at least two signals arerequired by T cells. The first one is the engagement of the T-cell receptor withpeptide-bearing MHC molecules. The other signal, termed costimulation, is receivedfrom a number of adhesion receptor-ligand interactions between the APC and T cell,and the recognition of B7 (including CD80 and CD86) and CD28 is the mostimportant one. The absence of costimulatory signal during T-cell recognition resultsin T-cell unresponsiveness. As an important costimulatory adhesion molecule, CD80is correlated with immune escape of tumor cells. Lacking of CD80, the second signalcannot initiated efficiently, tumor cells would escapehost immunity.The expression of costimulatory adhesion molecules such as CD80 on tumorsurfaces by gene transfer technique can create more immunogenic tumor cells whoinduce protective immunity against parental tumor challenge. Nevertheless, there arestill many shortages in gene transfection, such as difficulties of transferring primarycells, low efficiency, degradation of gene, oncogenicity after gene integration andtime consuming, which inhibit its clinical application.We may choose an alternative approach for the introduction of CD80 into thesurface of tumor cells. By using of molecular cloning technique, glycosyl-phosphatedylinositol and some proteins were gomphosis expressed. Fusion proteins can be anchored on cell surface membranes by GPI construction and are fullyfunctional. Proteins with GPI structure can modify cell surface efficiently. Thistechnology is called protein transfection, by which the surface protein composition ofcells can be manipulated without gene transfer. Such engineering offers severaladvantages over conventional gene transfer, such as 1) GPI-anchored proteins can beanchored onto cells which are difficult to transfect, 2) cells can be alteredimmediately without previous culturing, 3) the amount of protein added to the surfacecan be precisely controlled, and 4) multiple GPI-anchored proteins can besequentially or concurrently inserted into the same cells.In this article, in three parts we introduce the process of the construction ofhuman GPI-CD80 vector, expression fusion proteins in eukaryotic cells andtransferring GPI-anchored CD80 proteins into tumor cell membranes. The work is thefoundation for further research on tumor vaccine.Part 1 Derision and construction of eukaryotic expressing vector of humanGPI-CD80CD58 moleculars, also called LFA-3, in transmambrane and anchorage twoforms exist in cell membranes. CD58 gene of the latter form possesses GPI signalsequence, which could become the resource of GPI sequence for producing interestprotein. The extracellular domain of CD80/B7-1, has the ability to combine withCD28, then activate T cells.Objective:Designing and taking GPI signal sequence of human CD58, joining it withextracellular CD80 and constructing eukaryotic expressing vector pReceiver-M10/GPI-hCD80, which is necessary subject for further research.Methods: According to sequences of CD58 and CD80 provided by Genbank, primer P1and P2 were designed to amplify CD80 extracellular domain and primer P3 and P4 toamplify GPI signal sequence of CD58 by PCR. Two fragments were joined up byoverlapping PCR (SOE), then joined into T vectors, joined into eukaryotic expressionvector pReciever-M10 lastly. Recombinant plasmids were identified by doublerestriction enzyme digestion and were confirmed by sequencing.Results and conclusions:We successfully abtained the CD80 extracellular fragment and the GPI fragmentfrom Homo sapien CD58 by PCR. The sequencing result of the recombinant plasmidswas approximately coincident with the reference sequence provided by NCBI. Therecombinant plasmid was named pReceiver-M10/GPI-hCD80.Part 2 Transient expressing pReceiver-M10/GPI-hCD80 in COS-7, purifyingand identifying expression productCOS-7 is often used to transiently express a great quantity of proteins, by whichto assess whether the recombinant plasmid could express interest proteins ineukaryocyte, and to verify the eukaryotic expressing vector was constructed or not.Objective:To transient express pReceiver-M10/GPI-hCD80 in COS-7, to purify andidentify expression product, so as to prepare for constructing stable expression cellline.Methods:(1) Experiment group: purification of ultrapure, transfection grade recombinantplasmid DNA and transferring them into COS-7 by liposome. Transferred bypReceiver-M10 empty vector and not transfection COS-7 cells were as negativecontrol and blank respectively. To analyze the expression of CD80, detections on three groups of cells were performed as followed, indirect immunofluorescence, flowcytometry (FCM), SDS-PAGE, Western blot, extraction of total RNA and RT-PCR.(2) PI-PLC treatment: Cells were incubated with PI-PLC at 37℃for 1 hour, thencells were washed and analyzed for CD80 by FCM. Positive control, U251 cellstransfected by human CD80 confirmed; experiment group, negative control, andblank were same to (1).(3) Purification of fusion proteins: Extract total proteins of experiment group inlarge-scale and purify interest proteins by Ni-NTA agarose column. Save purifiedproteins for SDS-PAGE analysis.Results and conclusions:(1) Experiment group: IFL showed COS-7 sent out green fluorescence. FCMshowed 32.7％COS-7 cells are CD80~+. The SDS-PAGE results showed there was adeeply stained protein band at about 60 KD site. Western-blot analysis withmonoclonal antibody against human CD80 showed that the band could be stainedagain at the same site. AGE presented an evident band at about 840bp after RT-PCR.Control: All results were negative.(2) FCM assay results showed the fluorescence intensity of the cell transfectedby recombinant plasmids changed sharply before and after treated by PI-PLC, butthere were nearly no change in control cells.(3) SDS-PAGE showed purified proteins located at about 60 KD site.According to the experiment results above mentioned, we drew a conclusion thatCOS-7 cells transfected by recombinant plasmids pReceiver-M10/GPI-hCD80 couldexpress GPI-anchored human CD80.Part 3 Anchor GPI-CD80 proteins into cell surface membranes of U251 andprimary glioma cells Only if the fusion proteins expressed by COS-7 possessed GPI construction,they could be anchored into tumor cell membranes. As a tumor vaccine for clinicalapplication at the last destination, fusion protein GPI-hCD80 whether could beanchored on primary tumor cellular membrane is very important.Objective:To isolate primary glioma cells from the clinical operation sample and culturethem in vitro. To anchor GPI-CD80 proteins into cell surface membranes of U251and primary glioma cells and to prove the fusion proteins produced by COS-7 had theability of anchoring, preparing for produce proteins in large-scale for animalexperiment.Methods:Combining enzyme digestion and tissue culturing, we isolated and culturedprimary glioma cells. GPI-hCD80 fusion proteins and U251 and primary glioma cellswere co-incubated in 37℃for 4 hours then were observed the expression ofGPI-hCD80 by direct immuno fluorescence.Results and conclusions:Two kinds of tumor cells after anchored all showed red fluorescence underfluorescence microscope. The results imply that GPI-hCD80 fusion proteins wereanchored into tumor cells mambranes by GPI.SummaryWe successfully harvested CD80 extracellular fragment and the GPI fragmentfrom Homo sapien CD58, then constructed eukaryotic expression vector pReceiver-M10/GPI-hCD80. The GPI-CD80 fusion protein expressed by COS-7 was confirmedpossessing the capability of anchorage. All the work is the foundation for furtherstudy on the biologic activity of the fusion protein after anchored on the surface of tumor cells. In addition, the fusion protein as a kind of tumor vaccine whether couldmediate lethal effect on tumor cells is still unknown.