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题名: 抗对虾白斑综合症病毒(WSSV)单链抗体P1D3基因在毕赤酵母中的表达及其性质研究
作者: 杨毅
答辩日期: 2007-06-08
导师: 戴和平
授予单位: 中国科学院水生生物研究所
授予地点: 水生生物研究所
学位: 硕士
关键词: 对虾白斑综合症病毒(WSSV) ; 单链抗体 ; 中和抗体 ; 毕赤酵母 ; 活体中和试验
其他题名: Expression and Characterization of Single Chain Fragment Variable P1D3 Antibody against Shrimp White Spot Syndrome Virus in Pichia pastoris
摘要: 自1992年以来,对虾白斑综合症病毒(WSSV)已经在世界范围内给对虾养殖业造成了巨大损失。该病毒不仅对对虾具有高致病性,导致对虾7天内死亡率达100%,对许多其他水生生物也具有感染力,如桡足类动物,蟹类等。目前对该病还没有较好的治疗方法,只能以预防为主。 单链抗体(scFv)是由抗体的重链可变区(VH)和轻链可变区(VL)经一段柔软的短肽(linker)连接而成。它具有分子小,结构稳定,穿透性强 ,具备完整的抗原结合活性,很少或不会激起机体免疫应答等优点,并且单链抗体基因操作方便、可在各种宿主细胞中表达。这些优点使得单链抗体在疾病诊断和治疗方面具有较大的应用前景。 为了探索WSSV的致病机理,探索防治WSSV的新途径,我们研究小组构建了抗WSSV的单链抗体cDNA噬菌体展示文库,并从中获得了十几种特异识别WSSV的单链抗体。其中标号为P1D3的单链抗体,在原代对虾细胞培养实验中表现出有较高的中和病毒的能力。由此设想,能否用这个单链抗体通过被动免疫的途径,增强对虾抗WSSV的能力。 利用大肠杆菌表达系统制备抗体时,难免会将大肠杆菌引入到抗体中,而大肠杆菌对环境和对虾本身有一定的危害性。而酵母可以作为对虾饵料的成分,因此对对虾是安全的,且对环境没有显著危险。为了达到将单链抗体通过饵料途径引入对虾体内的目的,本实验尝试了将单链抗体P1D3在毕赤酵母中进行表达。本研究挑选到了单链抗体P1D3的高效酵母表达菌株,并对单链抗体基因在毕赤酵母中的表达条件进行了摸索,同时尝试了制备抗体添加饲料,并进行了活体中和试验。 结果表明,本研究成功地将单链抗体P1D3基因在毕赤酵母中以胞内和分泌两种方式进行了表达。其中分泌表达的抗体仍然具有很高的WSSV结合活性,而且活性远远高于胞内表达和大肠杆菌表达抗体的活性。表达条件优化后,抗体最高表达量达到302 mg/L。 通过皮下注射法和制备抗体饲料投喂法来研究抗体P1D3对WSSV的中和能力。研究结果表明,P1D3在对虾活体中,对WSSV没有明显的中和作用,分析原因可能是与抗体的浓度太低,抗体的稳定性较差有关,而且明显的中和作用也许需要多种抗体的协同作用。虽然本研究没有获得预想的结果,但仍然为将来的对虾活体病毒中和实验提供了基本实验方法和参考数据。
英文摘要: Since 1992, shrimp White Spot Syndrome Virus (WSSV) has caused great losses to the shrimp farming industry all over the world. WSSV can not only cause up to 100% mortality of shrimp within seven days , but also infect many other aquatic organisms, such as copepod, crabs. To date, there is no good method to cure this disease, therefore, the most important strategy is to prevent in advance. Single chain fragment variable (scFv) consists of the variable fragment of the heavy chain and the light chain of antibodies, with a soft linker between them. It remains the ability to bind to the antigen as the native antibody, and is small, stable, easy to penetrate but hardly arises immune response. Further more, it is easy for gene manipulation and can be expressed in many hosts. All these virtues make scFv have a great potential in diagnosis and therapy of diseases. In order to study the mechanism of WSSV infection, and a new way to prevent the disease, we constructed a phage-displayed scFv cDNA library against WSSV , and have got some scFvs specifically binding to WSSV. Among these scFvs, one scFv, designated as P1D3, could neutralize WSSV in the shrimp lymphoid primary cell culture. So, we think about the feasibility to enhance the ability of shrimp to resist WSSV by passive immunization with P1D3. When E. coli is used for the scFv expression, the scFv product may be contaminated by something of E.coli. E. coli is harmful to health of shrimp and environment. However, yeast is a kind of shrimp’s food and environmental friendly. In order to introduce scFv into shrimp body by feeding way, we tried to express P1D3 in Pichia pastoris. At last, we got a yeast colony that expressed P1D3 with high production. Simultaneously, we tried a neutralization test against WSSV with the yeast-expressed P1D3. The results showed that we succeeded in expressing P1D3 in Pichia pastoris, intracellularly and secretively. The P1D3 expressed secretively showed more activity binding to WSSV than the P1D3 expressed intracellularly in yeast or in E. coli. After optimizing inducing conditions, the production of P1D3 by yeast secretion could reach 302 mg/L. Neutralization test was carried out by intramuscular injection and oral way with P1D3 expressed by Pichia pastoris. The result showed that WSSV could not be neutralized evidently by scFv P1D3. The low density and instability of P1D3 may be related to this result. Otherwise, coaction of several antibodies may finally create an excellent neutralization of WSSV. Although we could not get a successeful result in this research, it still provided some basic methods and reference date for future research of virus neutralization in shrimp body.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12194
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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Recommended Citation:
抗对虾白斑综合症病毒(WSSV)单链抗体P1D3基因在毕赤酵母中的表达及其性质研究.杨毅[d].中国科学院水生生物研究所,2007.20-25
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