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Expression of the Recombinant Multi-epitope Gene of LMP2A of Epstein-Barrin Virus in Escherichia Coli, Preparation of Its Monoclonal Antibody and Application of the Protein in Serological Detection

Author: XuWenZuo
Tutor: Yao
School: Nanjing Medical University
Course: Microorganism
Keywords: EB virus Latent membrane protein 2A (LM2A) Recombinant gene Expression Monoclonal antibodies Nasopharyngeal carcinoma (NPC) Serological testing
CLC: R446.6
Type: Master's thesis
Year: 2007
Downloads: 140
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Abstract

Epstein-Barr virus (EBV) is a gamma herpes virus subfamily, widely disseminated throughout the world, carried by 95% of the adult population. EBV primary infection is usually caused by infectious mononucleosis disease (IM), once infected will carry for life. EBV can infect the human body resting B lymphocytes, the part of infected cells can be transformed into the lymphoblastoid cells (LCL) to establish latent infection. EBV carriers with a strong presence in the body of specific CTL, control of EBV latent infection, but can not get rid of the virus. Host immune dysfunction, unlimited proliferation of infected B cells transformed. Has confirmed EBV with Burkitt's lymphoma (BL), (HD), Hodgkin's lymphoma and nasopharyngeal carcinoma (NPC) is closely related. Nasopharyngeal belong to the EB virus caused one of the most common non-lymphocytic malignancy, 80% of the NPC cases worldwide occur in our country. View of nasopharyngeal not surgery, radiotherapy and chemotherapy can not effectively eliminate tumor cells, the vast majority of patients prone to distant metastases is very important, so early diagnosis and immunotherapy. EB virus is closely related to the occurrence of the NPC, through the detection of EBV antibodies for early diagnosis of NPC is an important means to improve the survival rate of patients with NPC. With the EBV serum epidemiological studies and the development of molecular biology, cloning, expression and purification the EBV of protein or polypeptide, and this antigen to create a simple, rapid, sensitive and specific method for detection of EBV antibodies is research the NPC diagnosis of the main development direction. EB virus latent membrane protein 2A (LMP2A) stable expression on the cell membrane of EBV-associated tumors latently infected with the virus and cell transformation is closely related to and involved in the regulation of transmembrane signal transduction function more and more attention. In this study, using the overlap PCR method contains the LMP2A four major epitopes of gene sequences, the recombinant gene named EC2A, prokaryotic expression plasmid pET32a-EC2A transformed into E. coli expression and purification; immunized with purified fusion protein mice to produce monoclonal antibodies and identification; preliminary application of the purified protein EB virus serological detection of patients with nasopharyngeal carcinoma. The main contents and results are as follows: a the EB virus LMP2A epitope recombinant gene expression in Escherichia coli and purified using software LMP2A gene sequence analysis, and predict its possible epitopes. The overlapping PCR gene contains the LMP2A four major antigenic epitope sequence, the the recombinant gene named EC2A. Construction of prokaryotic expression plasmid pET32a-EC2A. Recombinant plasmid was transformed into E. coli by PCR, restriction enzyme digestion, nucleic acid sequencing and analysis (Ecoli) BL21, induced expression of the fusion protein using nickel affinity chromatography purified by SDS-PAGE and Western blot results showed that the expression of the protein molecular weight approximately 40kD. EB virus LMP2A epitope fusion protein Preparation and identification of monoclonal antibodies to the purified fusion protein immunized mice, fusion in the conventional method, a stable secretion mouse anti-human LMP2A repeated cloning and subcloning The monoclonal antibody, named 1F5. The method of using ascites induced the production of monoclonal antibodies, after the preliminary purification of the antibody ascites by saturated ammonium sulfate precipitation method, a concentration of 10mg/ml. By rapid qualitative dipstick identification of the 1F5 heavy chain is IgG1, and the light-chain κ for. The ELISA assay showed that the purified monoclonal antibody titer was more than 1:10,000. Western blot analysis showed that the specificity of mAbs have good recognition LMP2A molecules. EB virus LMP2A epitope purified protein as antigen fusion protein in nasopharyngeal carcinoma serological testing, EB virus patients with nasopharyngeal carcinoma detected by ELISA in serum antibody, the results show a detection rate 84.4%. This method is compared with conventional VCA / IgA method sensitivity is slightly higher. This method is expected to add a method for early diagnosis of clinical NPC. In summary, this study contains the the LMP2A four major epitope gene sequence EC2A and expressed in E. coli, purified fusion protein was prepared by a monoclonal antibody was successfully cloned, and this protein is preliminary applied NPC serological testing of patients, can detect specific antibodies. This experiment has laid the foundation for future research LMP2A biological activity, this antigen is also expected to be used in clinical detection.

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