番鸭呼肠孤病毒MW9710株δC蛋白基因在毕赤酵母中表达

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基金项目:

福建省科技计划项目(2005N022);福建省科技计划重大专项(2006NZ003-2);福建省自然科学基金项目(2008J0061)

σC gene expresson of muscovy duck reovirus MW9710 strain in Pichia pastoris

1.
Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350013, China;
2.
Fujian Animal Diseases Control Technology Development Center, Fuzhou, Fujian 350013, China

摘要: 将番鸭呼肠孤病毒MW9710株的σC蛋白基因与毕赤酵母分泌性表达载体pPIC9K相连接,构建重组表达载体pPIC9Kσ-C,转化大肠杆菌DH5α,经PCR、酶切和测序鉴定,基因序列完全正确.纯化的重组质粒pPIC9Kσ-C用内切酶SacⅠ线性化,电转化毕赤酵母,使重组表达载体与酵母染色体发生同源整合,采用G418抗性梯度法筛选多拷贝重组菌株,用甲醇进行诱导表达,通过SDS-PAGE和Western-blot分析表达产物,结果表明目的蛋白得到了高效表达,并且具有免疫反应性.
- 番鸭呼肠孤病毒 /
- δC蛋白基因 /
- 毕赤酵母 /
- 表达
Abstract: The σC gene of DRVMW9710 was inserted into pPIC9K to construct a recombinant secretory vector,pPIC9K-σC.Then,pPIC9K-σC was transformed into E. coli DH5α.The recombinant vector was identified by PCR,enzymatic analysis and sequencing.The positive plasmid was linearized by Sac I and transformed into GS115 by electroporation.The insert was integrated into genomic DNA of Pichia pastoris by means of homologous recombination.The recombinants with multiple integrated copies of σC were screened using gradient concentration G418,and inducted with methanol.The results of SDS-PAGE and Western-blot showed that the highly expressed protein had an immunoreaction property.
- muscocy duck reovirus /
- σC gene /
- Pichia pastoris /
- expresson

[1]	欧阳岁东,胡奇林,林锋强,等.番鸭呼肠孤病毒MW9710株δC蛋白基因的克隆和序列分析[J].中国预防兽医学报,2006,28(5):510-513.
[2]	GALL-RECULE G L,CHERBONNEL M.Molecular characterization and expression of the S3 gene of muscovy duck reovirus strain 89026[J].J Gen Girology,1999,80:195-203.

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