| Epstein-Barr virus (EBV) is a ubiquitous humanγ-herpesvirus infecting approximately 95% of the adult population world-wide. The primary infection that causes infectious mononucleosis (IM) happens early in childhood and the infected individual will remain a lifelong carrier with latent infection. Importantly, EBV has the unique ability to transform resting B cells into permanent, latently infected lymphoblastoid cell lines (LCLs). Specific cytotoxic T cells (CTL) in the carriers control the latent infection but can not eliminate the viruses. When the patient's immune system is compromised, those infected B cells will be transformed immortal. It is proved that EBV, a definite DNA tumor virus, is associated with several malignant diseases including Burkitt's lymphoma (BL), Hodgkin's disease (HD), undifferentiated nasopharyngeal carcinoma (NPC). NPC derived from the epithelial cells occurs worldwide with variable incidence. NPC is a common human cancer with high incidence and almost 80% cases are in China. Because NPC is usually difficult to be removed by surgery, and the tumor cells could not be eliminated by conventional radiotherapeutic and chemotherapeutic means, relapse and metastasis of tumor will occur in some cases. Therefore, it is critically important to develop a rapid specific way to diagnosis NPC earlier and an immuno-therapeutic method.As EBV is associated with NPC, detection of its antibodies in the sera may be helpful in early diagnosis and improve the overall survival of patients. With the development of molecular biology and epidemiology of infection of human population, many EBV-specific proteins were expressed and purified which were used to detect antibodies in order to develop a rapid, sensitive, specific method for early diagnosis of NPC patients. Latent membrane Protein 2A (LMP2A) is characteristically expressed in latently infected B lymphocytes. This indicates that LMP2A plays an important role in viral latency and persistence through regulating trans-membrane signal transduction, which is drawing more and more attention.In this study, the recombinant multi-epitope gene containing four main LMP2A epitopes was synthesized by overlapping extension PCR and cloned into expression vector pET32a. The recombinant protein was expressed in Escherichia coli and purified to immunize Balb/c mice for preparation a monoclonal antibody. Also, it was used in serological detection of patients of NPC which is associated with EBV. The main results are as following:1. Expression and purification of the recombinant multi-epitope gene of LMP2A of Epstein-Barrin virus in Escherichia coliAfter analysis by the computer, the recombinant multi-epitope gene containing four main LMP2A epitopes named EC2A was synthesized by overlapping extension PCR and cloned into expression vector pET32a. The constructed vector which had been identified by PCR, enzyme digestion and nucleotide sequences analysis was transformed into BL21. After inducing with IPTG, the recombinant protein was purified with Ni~+ affinity chromatography. SDS-PAGE and Western blot analysis showed that the recombinant protein was about 40kD.2. Preparation and identification of a monoclonal antibody against the recombinant protein EC2ABalb/c mice were immunized with the purified protein EC2A and the splenocytes were fused with murine myeloma SP2/0 cells by the cell fusion hybridoma technique. By means of multiple cell subcloning and repeated screening, a hybridoma cell line (named as 1F5) stably secreting anti-EC2A monoclonal antibody was select out. Then the Ig isotypes were identified with test paper. The isotype of 1F5 was mouse IgG1. The ascites were produced in mouse abdominal cavity. The ascites were purified by precipitation with saturated ammonium sulfate and the titer of the antibody was over 1×10~4. Western blot analysis showed that 1F5 was bound to LMP2A protein specifically. 3. Application of the recombinant protein EC2A in serological detection of patients of NPCThe purified recombinant protein EC2A was used as an antigen to detect specific antibodies in the sera from NPC patients. 84.4% patients were positive for anti-EC2A IgG. Compared with anti-VCA IgA antibodies titers, we found that detection of LMP2A specific antibody could complement anti-VCA IgA test.In conclusion, we obtained the recombinant multi-epitope gene of LMP2A of EBV and it could be expressed with high performance in E.coli. We generated a monoclonal antibody recognizing LMP2A which may be used to study the biological activity of LMP2A. We also utilized the EC2A protein in serological detection of NPC patients which may be helpful in diagnosis and prognosis of EBV-associated malignancies, especially of NPC.
|
PDF Full Text Request