抗对虾白斑综合症病毒的单链抗体A1的表达和生物化学特性

用噬菌体展示技术制备了抗对虾白斑综合症病毒(WSSV)的单链抗体A1。该抗体在30℃培养条件下诱导表达20h后,其蛋白表达量可达总菌体蛋白的3.67%。用亲和层析柱和SephadexG-100层析柱可将单链抗体A1纯化为一条单电泳条带,其分子量约为31.5kD。用等电聚焦电泳测定,其等电点为pH5.8。ELISA测定表明冻干的单链抗体A1在室温储藏4年后与WSSV结合仍具有较高的活力。  

Studies on expression and biochemical characteristics of single chain fragment variable A1 against White Spot Syndrome Virus of shrimp

White Spot Syndrome Virus (WSSV)was first found in Taiwan in 1992.And since 1993,white spot syndrome disease of Shrimps caused by this virus was widely spread throughout China,Asia and pan-Pacific Ocean.It's a great harm to the aquaculture of shrimps.Electron microscopy studies show that the WSSV virion is an ellipsoid to bacilliform,enveloped,particle with a tail-like appendage at one end. The virion contains a rod-shaped nucleocapsid,typically measuring 275nm in length and 120nm in width.The WSSV virion has a large circular double-stranded DNA of about 290-305 kb.The International Committee on Taxonomy of Viruses approved a proposal to erect WSSV as the type species of the genus Whispovirus,family Nimaviridae(www.ncbi.nlm.nih.gov/ICTVdb/Ictv/index.htm).Our laboratory has constructed an antibody phage display library against WSSV.After four rounds of panning against WSSV,192 out of 480 clones displayed WSSV binding activity.One of the positive clones,designated A1,had relatively higher activity specifically binding to WSSV.In this study,we has prepared soluble scFv A1 and purified it.After induced to express at 30℃ for 20h,the amount of this antibody can reach to 3.67%of total proteins in the bacteria.Using affinity chromatography column and Sephadex G-100 column,scFv A1 can be purified to be a single band in the SDS-PAGE.Its molecular weight is about 31.5 KD.Using isoelectrofocusing electrophoresis,the isoelectric point of scFv A1 was determined to be pH 5.8.The tests of ELISA indicate that lyophilized scFv A1 stored at room temperature for 4 years still kept high activity to bind to WSSV.