仙台病毒Tianjin株噬菌体Fab抗体库的构建及初步筛选  

Construction and bio-panning of Fab phage display library against Sendai virus Tianjin strain

作　　者：李霞[1] 李晓绵[2] 石立莹[2] 李梅[2] 

机构地区：[1]牡丹江医学院,黑龙江牡丹江157011 [2]天津医科大学,天津300070

出　　处：《中国生物制品学杂志》2013年第4期482-485,共4页Chinese Journal of Biologicals

基　　金：国家自然科学基金(30471530)

摘　　要：目的构建仙台病毒Tianjin株噬菌体Fab抗体库,并进行初步筛选。方法用仙台病毒Tianjin株免疫小鼠,制备脾细胞悬液,提取小鼠脾细胞总RNA,RT-PCR扩增鼠抗体轻链(κ链)和重链Fd基因,将轻链基因和噬菌粒p3MH经SacⅠ和XbaⅠ双酶切后纯化回收,经T4 DNA连接酶连接,电转化E.coli XL1-Blue,构建轻链库;将重链Fd段基因和轻链库p3MH-κ经SpeⅠ和XhoⅠ双酶切后,纯化回收并连接,电转化E.coli XL1-Blue,获得噬菌体抗体库,计算重组率和抗体库滴度。以仙台病毒Tianjin株重组蛋白HN2为抗原进行初步筛选,制备噬菌体抗体,并进行测序。结果构建的免疫噬菌体抗体库库容为1.18×107,重组率为90%,制备的噬菌体抗体滴度为1011pfu/ml;经初步筛选,噬菌体富集了约63.6倍,获得2株与重组蛋白HN2有结合活性的阳性克隆,经NCBI BLAST进行同源性分析,显示为鼠源性抗体,经IMGT分析,显示轻链基因与Vk9亚群基因的同源性为94.87%,重链基因与VH7亚群基因的同源性为97.85%。结论成功构建了仙台病毒Tianjin株噬菌体Fab抗体库,为该病毒株的诊断、疾病治疗及致病机制等研究奠定了基础。Objective To construct and primarily screen the Fab phage display library against Sendai virus Tianjin strain.Methods Total RNA was extracted from spleen cells of mice vaccinated with Sendai virus Tianjin strain,with which antibody light chain（κ） and heavy chain（Fd） genes were amplified by RT-PCR.The κ gene and phagemid p3MH were digested with SacⅠand XbaⅠ,then purified,recovered,linked with T4 DNA ligase,and transformed to E.coli XL1-Blue by electroporation.The constructed light chain phage display library p3MH-κ and Fd gene were digested with SpeⅠ and XhoⅠ,then purified,recovered and linked,and transformed to E.coli XL1-Blue.The constructed phage antibody library was calculated for recombination rate and titer,and screened primarily using the recombinant protein HN2 of Sendai virus Tianjin strain as an antigen,and the obtained phage antibody was sequenced.Results The Fab phage antibody library against Sendai virus Tianjin strain,with a capacity of 1.18 × 107,was constructed,of which the recombination rate was 90%,and the titer of prepared phage antibody was 1011 pfu / ml.Primary screening proved that the phages were enriched by about 63.6 folds.Two positive clones with binding activities to recombinant HN2 protein were obtained,and proved as murine antibody by analysis of homology using NCBI BLAST software.IMGT analysis showed that the homology of light chain gene to Vk9 subgroup gene was 94.87%,while that of heavy chain gene to VH7 subgroup gene was 97.85%.Conclusion The Fab phage display library against Sendai virus Tianjin strain was successfully constructed,which laid a foundation of diagnosis of the strain,therapy of relevant disease as well as study on pathogenic mechanism.

关 键 词：仙台病毒 Tianjin株 FAB 噬菌体抗体库 

分 类 号：Q789[生物学—分子生物学]