鼠源抗烟曲霉单链抗体库制备及鉴定

发布时间：2018-01-03 12:09

本文关键词：鼠源抗烟曲霉单链抗体库制备及鉴定　出处：《中国公共卫生》2016年11期 　论文类型：期刊论文

【摘要】：目的构建鼠源抗曲霉菌单链抗体(sc Fv)噬菌体展示文库并进行初步鉴定,为制备用于侵袭性曲霉感染实验室诊断的特异性抗体提供新的方法。方法利用噬菌体展示技术构建鼠源抗烟曲霉半乳甘露聚糖(GM)Sc Fv库,经过4轮"吸附-洗脱-扩增"淘选富集,随机挑选克隆进行可变区基因测序确定文库多样性,并用phageELISA筛选烟曲霉特异性结合的克隆。结果构建的sc Fv噬菌体表达文库,库容量约为1.8×107;经4轮富集筛选,洗脱的表达sc Fv噬菌体滴度渐增;随机挑选出4轮筛选后的20个克隆,其可变区基因序列不同;随机挑选出4轮筛选后的90个克隆,其中6个克隆与烟曲霉GM特异性结合,而与对照的白假丝酵母菌无结合。结论构建并初步鉴定了抗曲霉菌单链抗体库,库容量大,VH和VL区具备多样性;筛选的sc Fv与曲霉菌GM特异性结合,提供了快速制备、筛选GM高特异性抗体分子的新方法。
[Abstract]:Objective to construct mouse anti-Aspergillus scFv phage display library and identify it preliminarily. Methods the mouse anti-Aspergillus fumigatus galactomannan (GMN) Sc FV library was constructed by phage display technique in order to prepare specific antibody for laboratory diagnosis of invasive Aspergillus fumigatus infection. After four rounds of "adsorption-eluation-amplification" enrichment, random selection of clones for variable region gene sequencing to determine the diversity of the library. The specific binding clones of Aspergillus fumigatus were screened by phageELISA. Results the Sc Fv phage expression library was constructed with a capacity of about 1.8 脳 10 ~ 7; After four rounds of enrichment and screening, the phage titers of eluting expression sc-Fv increased gradually. After 4 rounds of screening, 20 clones were randomly selected and their variable region gene sequences were different. 90 clones were randomly selected after 4 rounds of screening, of which 6 clones were specifically bound to Aspergillus fumigatus GM, but not to the control Candida albicans. Conclusion A single chain antibody library against Aspergillus fumigatus was constructed and preliminarily identified. The VH and VL regions of large reservoir capacity have diversity. The screened sc FV binds specifically to Aspergillus GM and provides a new method for rapid preparation and screening of GM highly specific antibody molecules.
【作者单位】：沈阳医学院基础医学院;辽宁省环境污染与微生态重点实验室;
【基金】：辽宁省科学事业公益研究基金(GY2014-A-007) 沈阳市科学技术项目计划(F14-181-1-00) 沈阳医学院科研立项项目(20141022)
【分类号】：R379
【正文快照】： Tw enty clones w ith different gene sequence of variable region w ere randomly selected after 4 rounds of panning.The di-versity of VH and VL w as confirmed in the single chain antibody library.Ninety clones w ere tested for binding specificityto Aspergi

【相似文献】