摘要
为利用原核表达方法使猪传染性胃肠炎病毒(TGEV)Sa蛋白表达在细菌的外表面,对克隆载体pMD18-T-pgsA进行KpnⅠ+BamHⅠ双酶切,将所得片段插入表达载体pET-32a(+)中,构建了重组质粒pET-pgsA,对克隆载体pMD18-T-Sa和重组质粒pET-pgsA分别进行BamHⅠ+HindⅢ双酶切,将Sa片段插入pET-pgsA中,构建重组质粒pET-pgsA-Sa。重组质粒pET-pgsA-Sa经PCR和序列鉴定为阳性后,转化入宿主菌大肠杆菌BL21(DE3)中,用IPTG进行诱导表达。SDS-PAGE分析结果表明,在70ku处出现了与预期大小相符的目的条带。Western-blot试验证实,融合基因pgsA-Sa获得了正确表达,具有良好的反应原性。间接免疫荧光试验证实融合蛋白在大肠杆菌表面获得了表达。结果表明,成功实现了TGEV Sa蛋白在细菌外表面的正确表达,初步鉴定其具有良好的反应原性。本研究为跨膜表达体系的建立和新型口服基因工程疫苗的研制奠定了基础。
To express the Sa protein of transmissible gastroenteritis virus of swine（TGEV） on the outer surface of bacteria by prokaryotic expression,the recombinant cloning plasmid pMD18-T-pgsA was digested with KpnⅠand BamHⅠ,and the pgsA fragment was purified and cloned into the KpnⅠand BamHⅠsites of plasmid pET-32a（＋） to construct a recombinant expression plasmid pET-pgsA.The recombinant cloning plasmid pMD18-T-Sa was digested with BamHⅠand HindⅢ,Sa fragment was purified and cloned into the BamHⅠand HindⅢ sites of plasmid pET-pgsA to construct a recombinant expression plasmid pET-pgsA-Sa.The recombinant plasmid pET-pgsA-Sa was verified by PCR and sequencing analysis and then transformed into Escherichia coli BL21（DE3） which was then induced with 1.0 mmol/L IPTG at 25 ℃.The expressed product with the size of 70 ku as expect was showed by SDS-PAGE.By analysis of Western-blotting,the expressed product can react with antibody to TGEV,indicating that fusion protein was expressed in the prokaryote system and showed good reactinogenicity.By indirect immunofluorescence assay,the successful display of fusion protein on the E.coli BL21 cell surface was tentatively confirmed.The TGEV Sa protein was correctly expressed on the outer surface of the bacteria,and had good reactinogenicity.The study laid a foundation for the creation of transmembrane expression system and development of new oral vaccine.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2011年第9期896-900,共5页
Chinese Veterinary Science
