Yu Xing-Long, Tu Chang-Chun, Xu Xing-Ran, Zhang Mao-Lin, Chen Yi-Xiang, Liu Bo-Hua
Changchun University of Agricultural and Animal Sciences, Changchun 130062, China.
Sheng Wu Gong Cheng Xue Bao. 2003 Jul;19(4):439-43.
Classical swine fever virus (CSFV), an enveloped positive-stranded RNA virus in the genus Pestivirus of the Flaviviridae family, is the causative agent of a highly contagious swine disease characterized by symptoms of hemorrhagic fever and immune depression, usually leading to substantial economic losses. The serological methods for detection of CSFV antibody such as ELISA are important means for the diagnosis of CSFV and immune surveillance. It is difficult to obtain CSFV antigen with high quality using traditional method because its titration titer is low in cell culture. CSFV has four structural protein named C, E0, El and E2. The E2 protein contains major antigenic determinants that are conserved between different CSFV strains and involved in neutralization by antibodies. So recombinant E2 protein can be developed as an alternative to the intact viral antigen. So far, CSFV E2 have not been expressed in E. coli with high level. Many factors, such as the secondary structure, the stability of 5' and 3' terminus of gene, the location of SD sequence and the bias of codes, are involved in the expressing level of foreign gene in E. coli . In this study, two sites of the E2 gene sequence were confirmed to be detrimental to its expression efficiency in E. coli through the computer-aided analysis. So they were mutated using recombinant PCR without changing the amino acids sequence of CSFV E2 gene. A plasmid was constructed by inserting the mutated E2 gene into the prokaryotic expression vector pET-28a(+) and named pETE2. The E. coli competent host BL21 (DE3)lysS transformed with pETE2 could express the E2 gene at high level, amounting to 28% of the total protein of the induced recombinant bacteria at the presence of IPTG. Except the hydrophobic transmembrane domain at C terminus, the recombinant E2 protein includes the total aa sequence. So it contains all the potential linear antigen epitopes of E2 protein because hydrophobic aa region can not form epitope. The recombinant E2 protein was CSFV-specific as proved by Western blotting and indirect ELISA. The rabbits immunized with the recombinant E2 can be protect from the challenge of hog cholera lapinized virus. This is the first report that E2 gene is expressed with high level expression in E. coli. In conclusion, it is an effective measure that mutate the CSFV E2 gene to increase its expression level in E. coli. The recombinant CSFV E2 protein possess fine immunonicity and can be used the antigen for the detection of CSFV antibody.
经典猪瘟病毒（CSFV）是黄病毒科瘟病毒属的一种有包膜的正链RNA病毒，是一种高度传染性猪病的病原体，其特征为出血热和免疫抑制症状，通常会导致重大经济损失。检测CSFV抗体的血清学方法如ELISA是诊断CSFV和免疫监测的重要手段。使用传统方法难以获得高质量的CSFV抗原，因为其在细胞培养中的滴定效价较低。CSFV有四种结构蛋白，分别命名为C、E0、E1和E2。E2蛋白包含主要抗原决定簇，这些决定簇在不同CSFV毒株之间保守，并参与抗体中和作用。因此重组E2蛋白可作为完整病毒抗原的替代品开发。到目前为止，CSFV E2尚未在大肠杆菌中高水平表达。许多因素，如二级结构、基因5'和3'末端的稳定性、SD序列的位置和密码子偏好性，都与外源基因在大肠杆菌中的表达水平有关。在本研究中，通过计算机辅助分析确定了E2基因序列的两个位点不利于其在大肠杆菌中的表达效率。因此，在不改变CSFV E2基因氨基酸序列的情况下，使用重组PCR对其进行了突变。通过将突变的E2基因插入原核表达载体pET-28a(+)构建了一个质粒，命名为pETE2。用pETE2转化的大肠杆菌感受态宿主BL21(DE3)lysS能够高水平表达E2基因，在IPTG存在的情况下，表达量占诱导重组菌总蛋白的28%。除了C末端的疏水跨膜结构域外，重组E2蛋白包含完整的氨基酸序列。因此它包含E2蛋白所有潜在的线性抗原表位，因为疏水性氨基酸区域不能形成表位。重组E2蛋白经Western印迹和间接ELISA证明具有CSFV特异性。用重组E2免疫的兔子可免受猪瘟兔化弱毒病毒的攻击。这是首次报道E2基因在大肠杆菌中高水平表达。总之，对CSFV E2基因进行突变以提高其在大肠杆菌中的表达水平是一种有效措施。重组CSFV E2蛋白具有良好的免疫原性，可作为检测CSFV抗体的抗原。
