EB病毒潜伏膜蛋白2A抗原表位的融合表达及其意义

为了探讨含有EB病毒潜伏膜蛋白2A(LMP2A)抗原表位片断表达的融合蛋白在鼻咽癌血清学检测中的应用意义,通过重叠延伸PCR方法,合成了3对相互重叠的寡核苷酸引物,涵盖LMP2A的主要的4个抗原表位,将它们拼接在一起构建一个多肽融合基因,克隆到PGEX-4T-2载体中表达融合蛋白,以GST亲和层析柱法纯化融合蛋白,鉴定后以此为抗原检测鼻咽癌患者的血清。结果表明,获得了含EB病毒LMP2A主要的4个抗原表位的融合蛋白(EC2A),蛋白纯度达90%以上,ELISA结果显示鼻咽癌患者血清的检出率为77.9%,正常人群血清为阴性,与常规的VCA-IgA法进行比较,有9份(13.3%)血清VCA-IgA为阴性而EC2A-IgG检出阳性,为鼻咽癌的临床检测提供了新思路,也为后续的单克隆抗体制备奠定了基础。

The fusion expression of EBV latent membrane protein-2A antigen epitopes and its significance

To explore the significance of fusion proteins of EBV latent membrane protein-2A (LMP2A) antigen in the serological diagnosis for nasopharyngeal carcinoma(NPC),3 pairs of oligonucleotide primers were designed according to the genomic sequences of 4 main antigenic epitopes, the fusion gene containing 4 main LMP2A epitopes was synthesized with overlapping extension PCR and then was cloned into vector pGEX-4T2 for fusion protein to express in E.coli. The recombinant protein was purified by GST chromatography column and this protein was used as antigen in ELISA in the serological diagnosis for NPC patients. In these ways,4 fusion proteins(EC2A) containing 4 main antigenic epitopes of EBV LMP2A antigens with the purities more than 90% were obtained. Tested with ELISA,it was demonstrated that 77.9% of NPC patients showed positive results in their sera,but negative results in health individual controls. Comparing with the anti-viral capsid antigen(VCA)-IgA and LMP2A-IgG titers in sera from 68 NPC patients,9 specimens of sera(13.3%)negative for anti-VCA-IgA were positive for LMP2A-IgG. It is evident that the recombinant fusion EC2A protein provides a suitable antigen for the detection of NPC patients as well as for the preparation of useful monoclonal antibodies.