猪瘟病毒E2基因的定点突变、在大肠杆菌中的高效表达及表达产物的免疫原性

Site-directed Mutagensis of the Major Antigen E2 Gene of CSFV, Its High Level Expression in Escherichia coli and the Immunonicity of Recombinant E2 Protein

用重组PCR技术对猪瘟病毒石门株E2基因进行了定点突变 ,然后将突变后的基因克隆至表达载体质粒pET 2 8a(+ )中 ,构建成重组质粒pETE2。将pETE2转入受体菌BL2 1(DE3 )plysS中 ,在IPTG的诱导下 ,重组转化菌可高效表达目的基因 ,表达量平均可达菌体蛋白总量的 2 8%。免疫印迹和间接ELISA表明所表达的蛋白是CSFV特异性的。

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, E1 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.