章晋勇, 汪建国, 李明, 顾泽茂, 龚晓宁. 圆形碘孢虫单链抗体库的筛选与阳性克隆特征分析[J]. 水生生物学报, 2008, 32(4): 568-578.
引用本文: 章晋勇, 汪建国, 李明, 顾泽茂, 龚晓宁. 圆形碘孢虫单链抗体库的筛选与阳性克隆特征分析[J]. 水生生物学报, 2008, 32(4): 568-578.
ZHANG Jin-Yong, WANG Jian-Guo, LI Ming, GU Ze-Mao, GONG Xiao-Ning. ISOLATION AND CHARACTERIZATION OF SPECIFIC CLONES REACTEDW ITH MY XOBOLUS ROTUNDUS RELATED ANTIGEN FROM A COMBINATORIAL PHAGE DISPLAYED SCFV L IBRARY[J]. ACTA HYDROBIOLOGICA SINICA, 2008, 32(4): 568-578.
Citation: ZHANG Jin-Yong, WANG Jian-Guo, LI Ming, GU Ze-Mao, GONG Xiao-Ning. ISOLATION AND CHARACTERIZATION OF SPECIFIC CLONES REACTEDW ITH MY XOBOLUS ROTUNDUS RELATED ANTIGEN FROM A COMBINATORIAL PHAGE DISPLAYED SCFV L IBRARY[J]. ACTA HYDROBIOLOGICA SINICA, 2008, 32(4): 568-578.

圆形碘孢虫单链抗体库的筛选与阳性克隆特征分析

ISOLATION AND CHARACTERIZATION OF SPECIFIC CLONES REACTEDW ITH MY XOBOLUS ROTUNDUS RELATED ANTIGEN FROM A COMBINATORIAL PHAGE DISPLAYED SCFV L IBRARY

  • 摘要: 噬菌体抗体库已成为单克隆抗体生成的重要技术之一。本文通过用圆形碘孢虫相关抗原对已构建的鼠源免疫噬菌体展示单链组合抗体文库进行生物淘选,并对几株相对亲和力较高的阳性单克隆序列特征、相对亲和力及热稳定性等进行了分析。ELISA、间接免疫荧光及免疫印迹等进行特征鉴定。结果表明,噬菌体抗体库技术对于分离抗圆形碘孢虫不同表位抗原是可行的,将为粘体动物的抗原物质及分布、寄生虫-宿主相互关系等研究提供丰富的抗体来源。

     

    Abstract: Polyclonal and monoclonal antibodies, especially the latter were extensively applied in diagnosis and treatment of fish diseases because of their hihg specificity and affinity.Hybridoma cells are pure sourch of monoclonal antibody (MAb) of desired specificity and present excellent promise in clinical implication.However, the potential have been mostly limited in theory since the establishment of hybridoma technology was made by Kohler&Milstein in 1975 for some intrinsic problematic limitations, especially Human Anti-Mouse Antibody responses(HAMA).In additional, hybridoma technology was labor·extensive and can not be satisfied with requirement of monoclonal antibodies in post-genomic era.The recombinant antibody or engineering antibody technology, especially phage display antibody library, developed in 1990 show great advantage over hybridroma technology in production of monoclonal antibodies.For parasitic diseases caused by Myxozoans, a worldwide constraint factor for development of aquaculture industry and many techniques based on the antibodies were developed to diagnose and studied the immunogenicity and antigenicity of different myxozoan species to find the candidate antigen molecules to control the severe diseases.Significant advances in our understanding of the Myxozoa have been achieved in recent years, however, diagnosis, prophylaxis and therapy of myxospomdiosi8 have not been thoroughly resolved.Myxobolus rotundus Nemezek, 1 9 1 1, is a serious pathogen for the farming of crucian carp, Carassius auratua (L.), allogynogenetic gibel carp and Carassius auratva gibelio(Bloch)in China.Establishment of practical early diagnosis strategy and searching the protective antigen molecules are vital to control the parasitic myxosporean disease.Two strains monoclonal antibodies were previously screened by hybridroma technology, but the low diversity and difficulty of storing limited their application. In the present work, four strains phage displayed monoclonal scFv with better affinity were isolated from previously constructed combinatorial pahge displayed seFv antibody library using M.rotundus related antigen.Their characters were analyzed by ELISA, dot-blot, western·blot, IFAT and immunohistochemi8try.The sequence analysis and alignment displayed that the heavy and light chain of four clones were originated from different mouse germline gene families.Among them, pCAN-6H could specially react with the soluble protein of M.rotundus spores and recognize antigen with a molecular weight of about 34kD.The antigens recognized by pCAN-6H were located in sporoplasm of M.rotundus spores.The pCAN. 3K could specially react with the surface antigen molecules of intact mature M.rotundus spores, especially in the openings of sutural ridge, but no response with polar capsule and sporoplasm.The molecular weight reacted by pCAN-3K ranged from about 17.6kD to 107kD.The pCAN-9A could specially react with the inner membrane of plasmodia.but no response with the middle and outer membrane which indicate probably the heterogenicity of plasmodia membrane and the antigens with molecular weight ranged from about 17.2 kD to 91 kD.The pCAN-3F could react with both antigens of the presporogonic stages and polar capsules which indicate the existence of species·specific antigen for M.rotundus.with molecular weight of about 40kD.

     

/

返回文章
返回