构建分泌型HBsAg单链抗体在毕赤酵母菌中的表达研究.pdfVIP

# 168 # 5 62003 23 3 HBsAg 1 2 2 2 2 1 1 1 熊 盛 , 任向荣, 唐永红, 粟宽源 , 余宙耀 , 罗 勇 , 王 一飞 , 李久香 ( 11 , 510632; 21 458 , 510640 ) : HBsAg (HBscFv ), pGEM-HBscFv HBscFv, P 1p astoris pPICZAA , P 1p astoris GS115KM71, PCRZeocin , , 3%~ 5%2 000 mg/L Zeocin ; , 32 000 HBscFv, 22%; HBscFv , HBsAg : ; ; ; : Q786 : A : 1001-2478(2003)03-0168-05 Construction of Secretory Single Chain Fv Antibody Against HBsAg Expressed in Pichia Pastoris 1 2 2 2 2 1 XIONG Sheng , REN Xiang-rong , TANG Yong-hong , SU Kuan-yuan , YU Zhou-yao , LUO Yong , WANG Yi- 1 1 fei , LI Jiu-xiang ( 11 Bio-Med ical Research and Develop ment Center , Jinan University , Guangz hou 510632, China 21 Center fo Inf ectious Diseases, The 458th H osp ital of PLA , Guangz hou 510640, China ) Abstract: To expressthe ant-iHBsAg single chain Fv (HBscFv ), the HBscFv, being amplified from plasmid pGEM- HBscFv was subcloned into expressionvector pPICZAA andthen transformed into Pichia p astoris GS115 and KM711 The recombinant Pichia strains, identifiedby direct PCR andZeocin-resistant screening of Pichia transformants, werecult-i vated and induced with methanol1 The target protein was determined by Western blotting and indirect ELISA1 It was found that highZeocin-resistant transformantswere3%~ 5% of the total transformants screened1 Thosetransformants in- duced could secrete HBscFvwith an apparent massweight of 32 kD into thecultural supernatants up to alevel of 22% of the total proteins. TheHBscFv produced in Pichia p astoris could berecognized specifically by aMcAb against theHB- scFv of the E 1coli origin andpossessed ability ofHBsAg-binding1 Key words: HBsAg; single chain Fv; Pichia p astoris