口蹄疫病毒结构蛋白VP1在谷氨酸棒状杆菌中的表达及优化

Expression and optimization of the structural protein VP1 of foot-and-mouth disease virus in Corynebacterium glutamicum

摘要:

【背景】口蹄疫(foot-and-mouth disease, FMD)是由口蹄疫病毒(foot-and-mouth disease virus, FMDV)引起的感染牛、羊和猪等偶蹄动物的主要疫病之一。口蹄疫病毒的结构蛋白VP1包含多个能够引起机体免疫反应的主要位点，因此VP1是研究亚单位疫苗的方向靶标。谷氨酸棒状杆菌 (Corynebacterium glutamicum)作为安全生产菌株，是医药用蛋白生产的优势细胞工厂。【目的】利用C. glutamicum作为受体菌株表达外源蛋白的优势实现VP1的外源表达。【方法】根据VP1结构蛋白的基因序列、相应功能和C. glutamicum的密码子偏好性设计并合成VP1基因，与pXMJ19载体连接构成重组质粒pXMJ19-VP1。C. glutamicum CGMCC 1.15647菌株用于表达VP1-6×his蛋白，并对蛋白表达元件启动子、5′非翻译端(5′UTR)、目的蛋白自身N端等进行优化，同时对培养条件等进一步优化，采用SDS-PAGE和Western blotting技术检测VP1蛋白的表达情况。最后应用间接酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA)测定本研究中生产的VP1的免疫活性。【结果】SDS-PAGE和Western blotting分析结果表明VP1蛋白能在C. glutamicum CGMCC 1.15647菌株成功表达，将P_tac启动子替换为合成型启动子P_H36能提高蛋白产量。在此基础上，通过插入不同的5′UTR序列和VP1蛋白N端氨基酸的改变能进一步提高蛋白产量并可利用CspB信号肽实现分泌表达。发酵试验表明，VP1摇瓶发酵培养最优条件为30 ℃、24 h。ELISA试验表明，本研究中的VP1可与阳性血清特异性结合。【结论】本研究在谷氨酸棒状杆菌中成功表达FMDV的VP1蛋白，并通过优化蛋白表达元件的方法进一步提高了产量，为开发新型FMD免疫诊断试剂和安全高效的亚单位疫苗奠定了良好的基础。

Abstract:

[Background] Foot-and-mouth disease (FMD) caused by foot-and-mouth disease virus (FMDV) is one of the major diseases that infect cloven-hoofed animals such as cattle, sheep, and pigs. The structural protein VP1 of FMDV contains multiple sites that can cause host immune response, serving as a target for the research and development of subunit vaccines. As a safe production strain, Corynebacterium glutamicum is an elite cell factory for the production of pharmaceutical proteins. [Objective] To realize the heterologous expression of VP1 in C. glutamicum. [Methods] According to the gene sequence and function of VP1 and the codon preference of C. glutamicum, we designed and synthesized the VP1 gene and then ligated it with pXMJ19 to create the recombinant plasmid pXMJ19-VP1. C. glutamicum CGMCC 1.15647 was used to express VP1 protein. The protein expression elements [e.g., the promoter, 5′ untranslated region (5′UTR), and N-terminus] of VP1 protein and the culture conditions were optimized. SDS-PAGE and Western blotting were employed to determine the expression of VP1. Indirect enzyme-linked immunosorbent assay (ELISA) was employed to measure the immunocompetence of VP1 produced in this study. [Results] SDS-PAGE and Western blotting showed that VP1 was successfully expressed in C. glutamicum CGMCC 1.15647. Replacing P_tac promoter with the synthetic promoter P_H36 increased the protein yield. On this basis, the protein yield can be further increased by insertion of the 5'UTR sequence and mutation of the N-terminal amino acid of VP1. CspB signal peptide can be used to achieve secretory expression. Fermentation experiments showed that the optimum conditions of shake flask fermentation were 30 ℃ and 24 h. The results of ELISA suggested that VP1 could specifically bind to the positive serum. [Conclusion] The VP1 protein of FMDV was successfully expressed in C. glutamicum CGMCC 1.15647, and optimizing protein expression elements increased the protein yield, which laid a foundation for the development of immunodiagnostic reagents and safe and efficient subunit vaccines for FMD.