摘要
将RHDVVP6 0基因插入酵母转移载体pPICZB中转化毕赤酵母菌GS115株 ,经筛选获得染色体基因组中整合入VP6 0基因的重组酵母菌。以甲醇诱导培养后经SDS PAGE和Westernblot检测表达产物 ,在 6 0kD处出现一特异蛋白条带 ,表明RHDV的衣壳蛋白得到了成功表达。血凝试验表明 ,表达的重组蛋白具有血凝特性 ,可以凝集人“O”型红细胞 ,血凝价达 2 8,同时 ,该血凝性可被抗RHDV的高免血清所抑制。经电镜观察 ,重组酵母表达的衣壳蛋白可以在酵母菌体内自聚成大小约4 0nm ,和天然RHDV病毒子在物理形态上类似的病毒样颗粒 (VLPs)。该病毒样颗粒与兔抗RHDV高免血清作用后可被凝集成团 ,表明该VLPs与天然RHDV在抗原性上也极为相似。
The capsid protein (VP60) gene of RHDV was subcloned into the Pichia expressin vector pPICZ B to express the VP60 protein intracellularly. The recombinant plasmid was initially transformed into a E. coli strain TOP10 F′. After verification of the construct by sequencing, the recombinant plasmid was linearized by Sac I in the 5′ AOX1 region and then transformed into Pichia pastoris strain GS115 using the Pichia EasyComp Kit. After selecting and verifing for the insertion of VP60 gene in the genome, two clones of Pichia transformants were select for expression test. The recombinant clones were first inoculate with BMGY in baffled flask at 28～30℃ in a shaking incubator (250～300 r/min) until culture reaches an OD 600 = 2～6, then resuspend the cell pellet to an OD 600 of 1 0 in BMMY medium to induce expression for 5 days by methanol at a concentration of 0 5% in a 1 liter baffled flask covered with 2 layers of sterile gauze. Collect the cell pellets and break it by acid_washed 0 5 mm glass beads. The expression of recombinant Pichia strains was detected by SDS_PAGE and Western analysis with a polyclonal serum which showed a specific protein band of 60kD. Theses results indicates that the recombinant VP60 produced in Pichia was antigenically similar to the viral polypeptide. Electron microscopic observation of the recombinant Pichia _derived protein revealed the presence of virus_like particles similar in size and appearance to native virus capsids. In the haemagglutination test, the recombinant VLPs, like the native RHDV, also agglutinated human blood type O erythrocytes and could be inhibited by the anti_RHDV polyclonal serum.
出处
《生物工程学报》
CAS
CSCD
北大核心
2005年第1期135-138,共4页
Chinese Journal of Biotechnology
基金
国家自然科学基金项目资助 (No .3 0 40 0 3 2 0 )。~~
关键词
兔出血症病毒
衣壳蛋白基因
毕赤酵母
表达
病毒样颗粒
Rabbit haemorrhagic disease virus, capsid protein gene, expression, Pichia pastoris , VLPs
