【作者】 徐永春；

【导师】 张林；

【作者基本信息】 四川大学 ， 法医学， 2005， 硕士

【摘要】 [背景和目的] EB病毒(Epstein-Barr virus,EBV)是γ疱疹病毒的一种,与鼻咽癌(nasopharyngeal carcinoma,NPC)、霍奇金氏病(Hodgkin’s disease,HD)、等多种恶性肿瘤密切相关,被国际癌症研究中心列为病毒性致瘤因子。EB病毒的生活周期可分为潜伏期和裂解期两个阶段,其中裂解期又可细分为立即早期、早期、病毒DNA复制期和晚期。Rta蛋白是EB病毒裂解立即早期基因BRLF1的编码产物,是EB病毒由潜伏期转向裂解期的关键性调控因子之一。它能引起一系列裂解早期基因的相继表达,最终引发EB病毒裂解感染。此外,Rta蛋白和细胞因子之间的相互作用在调节宿主细胞分化增殖方面也发挥了重要的作用。本研究旨在制各针对EB病毒Rta蛋白的高效价和特异性的多克隆抗体,并初步应用于EB病毒相关肿瘤组织中Rta蛋白表达情况的研究。 [方法] (1)将已构建的两个原核表达质粒pGEX-R185I和pGEX—R150I更多还原

【Abstract】 [Background and Objective]Epstein-Barr virus (EBV) is a lymphotropic human gamma herpesvirus.There are several human cancers that closely associated with EBV infection, such as nasopharyngeal carcinoma (NPC) and Hodgkin’s disease (HD). EBV is one of the factors that has oncogenicity, definited by the International Agency for Research on Cancer (IARC). Protein Rta (BRLF1 transcription activator) is the gene product of BRLF1, which is one kind of the immediate-early gene (IE gene) in EBV lytic cycle. Rta is also one of the most important activators that regulates the EBV from latency to lytic cycle , its expression can lead to the activation of early genes and viral replication. Now Rta is the emphasis of reseach in the world, but there were few reports on any antibody to Rta. The aim of this research is to prepare the specific polyclonal antibody against protein Rta and study its expression in EB virus correlated diseases.[Methods]Two recombinant plasmids pGEX-R185I and pGEX-R.150I had been constructed in prior research. They were transformed into Escherichia coli BL21(DE3), respectively. Expressions of the two recombinant proteins GST-Rta185 and GST-Rta150 were induced by 0.1 mmol/L IPTG for 3.5h in LB medium(including Amp).The proteins,from lysates,were purified with Glutathione Sepharose 4B. SDS-PAGE was used to analyse the efficiency of the proteins’ expression and the purity of proteins.Each purified protein was mixed with Freund’s adjuvant and then immunized the rabbits respectively. After fundamental immunity and strengthening immunity, the serums of rabbits were isolated. The titer and the specificity of the antibodies were analyzed by ELISA and Western blot. The two crude antibodies were then purified through the sedimentation of the saturated ammonium sulfate (SAS).The anti-GST elements in the antibodies were removed by affinity chromatography. The efficiency of the purification was analyzed by ELISA and Western blot.The two antibodies were then used to detect the expression of the protein Rta in 30 cases of NPC and 15 cases of HD samples through the method of immunohistochemistry (Envision two-step ways).Another 30 cases of normal nasopharyngeal tissues and 15 cases of lymphatic tissues near to the tumor were also detected. [Results]The results of SDS-PAGE analysis suggested that a high level expression of target proteins was detected in the lysates and the ultrasonic treated supernatants. The two purified proteins GST-Rta185 andGST-Rta150 were obtained through the method of affinity chromatography with Glutathione Sepharose 4B.The results of Western blot and ELISA analysis suggested that the two crude polyclonal antibodies, anti-Rta185 and anti-Rta150, were specific, and their titers were all more than 1:32000. After purification of two antiserums, the purity of the antibodies was improved and the titer were not affected obviously , the elements of anti-GST had been removed efficiently.In the immunohistochemical analysis, about 73.3%(22/30) cases of NPC and 33.3%(5/15) cases of Hodgkin’s disease samples showed positive response to anti-Rtal85, while 15.0%(3/30) and 13.3% (2/15) cases of corresponding normal tissures were positive.At the same time, about 33.3 % (10/30) cases of NPC and 26.7% (4/15) cases of HD showed positive response to anti-Rta150, while 20.0 % (6/30)and 13.3% (6/30) cases of corresponding normal tissures were positive. In addition, 20% (6/30) cases of NPC and 13.3 % (2/15) cases of HD showed positive response to both two antibodies. [Conclusion]The prepared two polyclonal antibodies anti-Rta185 and anti-Rta150 have good specificity and high titer. The results of immunohistochemistry suggest that protein Rta expressed both in NPC and in Hodgkin’s disease, but the positive rate of Rta expression in NPC is more high. The preparation of two polyclonal antibodies are important for the research of protein Rta and the diagnosis or treatment of EB virus correlated diseases.更多还原