| Epstein-Barr Virus (EBV) is a human herpesvirus that establishes a lifelong latent infection in B lymphocytes. EBV is associated with the development of several malignancies of lymphoid origin, including Burkitt's lymphoma, Hodgkin's disease, and lymphoproliferative disorders arising in immunocompromised patients. There are also diseases of epithelial origin associated with EBV infection, including nasopharyngeal carcinoma and oral hairy leucoplakia.Nasopharyngeal carcinoma derived from the epithelial cells which which is prevalent in Southeast Asia, North Africa and in certain indigenous populations of North America, NPC is a common human cancer with high incidence . NPC is usually difficult to be removed by surgery ,and the tumor cells also could not be eliminated by conventional radiotherapeutic and chemotherapeutic means, relapse will occur in most NPC cases .Therefore, it is critically important to develop an rapid specific way to diagnosis NPC to improve livability.Viral Epstein-Barr nuclear antigen 1 (EBNA1), EBV-encoded RNAs (EBERs), latent membrane protein 1 (LMP1) and LMP2A transcriptshave been detected in tumour cells from patients with EBV-associatedmalignancies such as NPC and HD. LMP2A is one of the mostconsistently identified in vivo in B cells from healthy individualsharbouring a latent EBV infection This indicates that LMP2A plays animportant role in viral latency and persistence, which is necessary for thedevelopment of EBV-associated diseases. Recently more and moreevidence demonstrates LMP-2A induces significant humoral immuneresponses in EBV-associated malignant conditions especially innasopharyngeal carcinoma (NPC) patients whose sera are almost positivefor these Abs, and their liters correlate with the clinical condition of thetumors. LMP-2A specific cellular immune responses have also been welldocumented. The detection of these Abs may be helpful not only in thediagnosis and prognosis of EBV-associated diseases but may also provideinsights about their pathogenetic processes. We report here our studies onthe detection, analysis, and significance of LMP-specific Abs in differentEBV-associated diseases and show for the first time that these Abs canreadily be demonstrated in the sera of patients with EBV-associatedmalignancies and that anti-LMP may be of prognostic value in patientswith advanced stages of NPC.In the first part, digestion of pPICC/LMP2A with EcoR I, Not I released the 1500bp LMP2A fragment which was thereafter subcloned into the same restriction sites of the pPICZaA yeast expression vector,which carries a factor secretion signal sequence and with a Zeocine maker for antibiotic selection . The sequence of the amplified cDNA fragment was identical to that of EBV prototype B95.8 strain previously reported. This construct was designated pPICZaA/LMP2A.In the second part , pPICZoA/LMP2A was linearized with Sac I followed by transformation of Pichia Pastoris GS115. Positive recombinants were picked out by plating strains on YPG/Zeocine plates and sequentially were induced for LMP2A expression by culturing them in BMGY medium, followed in BMMY medium under the induction of 1% methanol. The target protein in supernatant of yeast culture was detected by 12% SDS-PAGE, weastem blotting. The target protein of culture supernatant is 20ug/ml with a 54 KDa molecular weight.In the third part, for improving the production of LMP2A protein, the automatically controlled fermentator was used. Pichia Pastoris GS115 grows well in the defined basal salts medium for fermentation. To purify the recombinant soluble LMP2A, the supernatant was harvested and dialysate overnight 4C,against 20 mmol/L of Tris-HCl buffer, PH =8.0. The dialysate was loaded onto a Q-Sepharose column (PH=8.0) and FPLC was performed.In the last part, the purified recombinant LMP2A was used as an antigen to Analysis of Anti-Latent Membrane Protein 2A Antibodies in the Sera of Patients with EBV-Associated Diseases. These studiesdemonstrate, for th...
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