| Nasopharyngeal carcinoma is one of the high incidence and malignant tumors in China,with the highest incidence of malignant tumor in the throat area.Its incidence rate in southern China and parts of southeast Asia and Africa is much higher than that in developed countries and regions such as Europe and America.The anatomical structure of the nosepharyngeal cancer is intricate,often not suitable for surgery,but the radiation sensitivity of the early nasal carcinoma is good,so the clinical preferred treatment is radiotherapy.Radiotherapy is effective in the treatment of early nasopharyngeal carcinoma.However,due to the limitations of early screening,most of the diagnosed patients are in the middle and late stage of clinical and have distal metastasis.In addition,some patients relapse after treatment,and in the case of late-stage,metastatic,or recurrent nasal cancer,the tumor tissue had high heterogeneity and decreased sensitivity to radiation,and the prognosis was low and the five-year survival rate was significantly less.In addition,the side effects and complications caused by radiotherapy greatly reduce the quality of life of patients.Therefore,it is increasingly important to find and explore biological therapeutic targets for the treatment of nasopharyngeal carcinoma.Nasopharyngeal carcinoma is closely associated with EBV.In NPC tissues,EBV exists in the state of latent II,expressing two important EBV encoded membrane protein LMP1 and LMP2,which is considered as important target proteins in the diagnosis and therapy of NPC.This research proposed two EBV LMP1 and LMP2 coding membrane proteins as targets to explore the screening strategy for specific diagnosis and treatment mAb of NPC on the monoclonal antibody screening platform in mice and rabbits.The results of this study can provide references for immune screening of specific antibody in outer region of transmembrane proteins.At the same time,the rabbit monoclonal antibody screening platform stablished in this study is of great significance for the development of more valuable antibodies for diagnosis and treatment of pathogenic microorganisms or tumors,with good expansibility and portability.The first part of this paper aims to explore the screening strategy of specific diagnosis and treatment of LMPs related nasopharyngeal carcinoma using the monoclonal antibody screening platform in mice.First,by analyzing the protein sequence of LMPs,the amino acid sequences of LMP1 and LMP2 in the extracellular region were determined,and various forms of immunogen such as synthetic peptide,prokaryotic tandem recombinant expression,eukaryotic expression of insect cells and whole cells were synthesized and prepared.In combination with Freund's adjuvant,aluminum adjuvant,CpG adjuvant,polysaccharide,small molecule agonist and other adjuvants,various immunoprotocols were designed to perform mouse immunization.and outcomes of different immunological schemes were evaluated through ELISA and band Western Blot with membrane protein expressing cells.It was found that that solubility of the synthetic peptide antigen is poor and the specific antibody can hardly be induce.Extracellular antigen is difficult to express in soluble form in E.coli.The full-length antigens of LMP1 and LMP2A expressed by insect cells are difficult to be purified.No difference was observed between different immunological regimens or adjuvant groups in inducing extracellular specific immunity.Secondly,different screening methodswere compared,including flow cytometry based on polypeptides,cell ELISA,ELISPOT,ELISA based on membrane protein or whole cell lysis.It was found that the specific b-cell separationbased on polypeptides as antigens has a poor specificity,and cell ELISA was the most suitable screening strategy for high-throughput screening of cell membrane antigens.In the end,we did a series of qualitative and applied research on the selected 97 antibodies.It turns out that there was no specific recognition of the antibodies in the outer regions of the LMPs,and there were four LMP1 cells that had a good natural antigen binding ability,and the epitopes they recognized were reported for the first time.Detection in mice xenograft and clinical biopsy of NPC patients found that anti-LMP1 mAb 9F2 and anti-LMP2 mAb 3A7 has good immunohistochemical reactive specificity,which are promising candidates for the histologic diagnosis of nasopharyngeal carcinoma clinical.In addition,mAb 9F2 could significantly reduce the level of NF-?B activity in LMP1 positive cells when transfected,but had no significant influence on the growth and metastasis of cells.The second part of this study aims to establish an efficient rabbit monoclonal antibody screening platform and explore its application in LMPs related specific antibody screening.First,the FCM-based sorting scheme of rabbit b-cell population was determined.Rabbit single B cell PCR was successfully constructed by a nested PCR of antibody variable region using specific primers,and the rate of the mAb genes were amplified successfully at a rate between 60%and 100%.Secondly,an efficient monoclonal antibody screening platform for rabbit B cells stimulated in vitro was successfully established.The optimal in vitro culture conditions of rabbit B cells were established.Under this condition,the positive rate of B cells in vitro culture was 38.75%on average(OD value>0.5).PRVRCH/pRVRCL,an external recombinant expression vector of rabbit monoantibody,was successfully constructed to express rabbit monoclonal antibody in hek-293 eukaryotic expression system.This technology platform was successfully used in the screening of rabbit monoclonal antibodies specific to human hepatitis E and rotavirus,respectively,confirming that the rabbit monoclonal antibody platform is feasible..Finally,the LMPs specific antibody was preliminarily explored using rabbit monoclonal antibody platform.No significant LMP1 specificity was observed in the Western Blot detection of antiserum in rabbits immunized with GST-LMP1-EMD and LMP1 expressing cells Significant LMP2A specificity can be observed in the blood of rabbits immunized with 2A-N.More attempts at immunization programmes remain to be explored.To sum up,this thesis aimed at exploring strategies of screening antibodies specificly binding the EBV latent membrane protein LMP1 and LMP2 intracellular or extracellular region.It turns out it's difficult to induce antibodies specific to extracellular membrane in mice.An efficient platform for the rabbit monoclonal antibody screening and expression were establish,which laid a foundation for the development of the specific antibody of diagnosis and treatment of nasopharyngeal carcinoma. |
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