抗犬瘟热病毒噬菌体抗体库的构建  

Construction of anti-canine distemper virus phage display antibody library

作　　者：马云青[1] 任文陟[1,2] 高娟[1] 陈健[1,2] 岳媛[1] 高红博[1] 高巍[1,2] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062 [2]吉林大学农学部实验动物中心,吉林长春130062

出　　处：《中国生物制品学杂志》2013年第6期813-816,共4页Chinese Journal of Biologicals

基　　金：吉林省科技发展计划青年科研基金(201201014);吉林大学基本科研业务费项目(450060481144);吉林大学农学部引进人才科研启动基金(4305050102Q7);吉林大学国家级"大学生创新性实验计划"(2011A81141)

摘　　要：目的构建抗犬瘟热病毒(canine distemper virus,CDV)T7噬菌体单链抗体(single-chain antibody fragment,scFv)库。方法用灭活CDV经皮下注射免疫犬,共3次,每次间隔2周,第3次免疫后2周采集犬外周血,分离淋巴细胞,提取淋巴细胞总RNA,RT-PCR分别扩增犬IgG重链(heavy chain,VH)和轻链(light chain,VL)基因片段,通过SOE-PCR法将VH和VL用一段linker连接组装成scFv基因,经EcoRⅠ和HindⅢ双酶切后,连接到表达载体T7 Select 10-3b上,用包装蛋白包装后,以大肠埃希菌BLT5403为宿主菌进行增殖,随机挑取50个噬菌斑,进行PCR鉴定,并测定文库滴度。结果扩增的VH和VL基因大小均与预期相符,测序结果经blast比对,扩增的片段与犬IgG的VH和VL序列匹配率达90%以上;所构建的原始文库滴度为3.2×107pfu/ml,扩增后的文库滴度为5.0×108pfu/ml。对随机挑取的50个噬菌斑进行PCR鉴定,重组率为94%。结论成功构建了抗CDV的T7噬菌体抗体库,为犬科动物犬瘟热的防治提供了参考。Objective To construct the T7 phage single-chain antibody fragment（scFv）library of anti-canine distemper virus（CDV）.Methods Dogs were immunized s.c.with inactivated CDV for 3 times each at an interval of 2 weeks,of which the peripheral blood samples were collected 2 weeks after the 3rd immunization.Lymphocytes were isolated,from which total RNA was extracted for amplification of heavy chain（VH） and light chain（VL） of IgG by RT-PCR and spliced by SOE-PCR using a linker.The obtained scFv gene was digested with EcoRⅠ and Hind Ⅲ,inserted into expression vector T7 Select 10-3b,packaged by packaging protein,and transformed to E.coli BLT5403 for proliferation.Fifty plaques were randomly selected and identified by PCR,based on which the library was tested for titer.Results Both the lengths of amplified VH and VL genes were consistent with those expected,of which the coincidence rate of sequence to those of canine IgG were more than 90% as proved by blast software.The titer of constructed primary library was 3.2 × 107 pfu/ml,which reached 5.0 × 108 pfu/ml after proliferation.The recombination rate of fifty plaques was 94% as proved by PCR.Conclusion The T7 phage single-chain antibody fragment library of anti-CDV was successfully constructed,which provided a reference for prevention and treatment of canine distemper.

关 键 词：犬瘟热病毒 单链抗体 噬菌体展示文库 

分 类 号：Q939.94[生物学—微生物学] R392.33[医药卫生—免疫学]