Expression of the recombinant extracellular gene of LMP2A of Epstein-Barr virus in escherichia coli and its application in serological detection
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摘要:
目的：构建EB病毒潜伏膜蛋白2A（LMP2A）胞外区重组基因的表达载体在大肠杆菌中表达，并将表达的蛋白用于EB病毒相关鼻咽癌（NPC）患者的血清学检测。方法：用BamHⅠ和EcoRⅠ将含LMP2A胞外区重组基因的质粒pEC2A双酶切后,克隆到pET32a中。重组质粒经PCR、酶切鉴定、核酸序列分析后转化大肠杆菌（E.coli）BL21，以异丙基硫代-β-D-半乳糖苷（IPTG）诱导表达融合蛋白，表达的蛋白经镍柱亲和层析纯化，并用Western blot法检测其抗原活性。用纯化的蛋白进行鼻咽癌患者血清学检测。结果： 重组表达质粒经PCR及酶切鉴定为阳性，核酸序列分析正确。SDS-PAGE和Western blot 结果显示表达的重组蛋白分子质量约为40 ku。以此为抗原能在鼻咽癌患者血清中检测到特异性的抗体。结论：成功获得了LMP2A胞外区重组基因表达的蛋白，纯化的蛋白可用于EBV相关鼻咽癌患者的血清学检测。
Abstract:
Objective：To construct a plasmid pET32a-EC2A for the expression of recombinant extracellular gene of LMP2A of Epstein-Barrin virus（EBV） in escherichia coli（E.coli） and utilize EC2A protein in serological detection for patients with nasopharyngeal carcinoma（NPC）. Methods：The plasmid pEC2A containing the recombinant extracellular gene of LMP2A of EBV was digested with BamHⅠ and EcoRⅠ and cloned into the expression vector pET32a. The constructed vector which had been identified by PCR, enzyme digestion and nucleotide sequences analysis was transformed into BL21. After induced with IPTG, the recombinant protein was purified with Ni+ affinity chromatography, at the same time, its antigenic activity was identified by Western blot. The purified protein was used in serological detection for patients with NPC which is associated with EBV. Results：The recombinant plasmid was constructed correctly. SDS-PAGE and Western blot analysis showed that the recombinant protein was about 40 ku. The purified protein could be used as an antigen to detect specific antibodies in the sera of patients with NPC. Conclusion：The recombinant extracellular gene of LMP2A of EBV could be expressed with high performance and the purified protein may be used in serological detection of antibodies for patients with NPC.
