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中华卫生应急电子杂志 ›› 2021, Vol. 07 ›› Issue (05) : 287 -291. doi: 10.3877/cma.j.issn.2095-9133.2021.05.007

论著

创伤弧菌溶细胞素噬菌体单链抗体库的构建
潘振1, 陈杰敏1, 胡成进2,()   
  1. 1. 213000 江苏常州,南京医科大学附属常州第二人民医院检验科
    2. 250031 山东济南,中国人民解放军第960医院实验诊断科
  • 收稿日期:2021-06-22 出版日期:2021-10-18
  • 通信作者: 胡成进
  • 基金资助:
    全军医学科技"十二五"重点项目(BWS12J014)

Construction of mouse-derived phage display library of scFv fragments against Vibrio vulnificus VvhA

Zhen Pan1, Jiemin Chen1, Chengjin Hu2,()   

  1. 1. Department of Clinical Laboratory, The Affiliated Changzhou No.2 People’s Hospital of Nanjing Medical University, Changzhou 213000, China
    2. Department of Laboratory Medicine, 960 Hospital of PLA, Jinan 250031, China
  • Received:2021-06-22 Published:2021-10-18
  • Corresponding author: Chengjin Hu
引用本文:

潘振, 陈杰敏, 胡成进. 创伤弧菌溶细胞素噬菌体单链抗体库的构建[J]. 中华卫生应急电子杂志, 2021, 07(05): 287-291.

Zhen Pan, Jiemin Chen, Chengjin Hu. Construction of mouse-derived phage display library of scFv fragments against Vibrio vulnificus VvhA[J]. Chinese Journal of Hygiene Rescue(Electronic Edition), 2021, 07(05): 287-291.

目的

为获得高特异性创伤弧菌溶细胞素单链抗体,利用噬菌体展示技术建立鼠源性噬菌体单链抗体库。

方法

用溶细胞素蛋白免疫BALB/c小鼠后,取脾脏提取总RNA,逆转录获得cDNA。以cDNA为模板扩增出抗体的轻链和重链可变区基因片段,通过OE-PCR技术拼接成完整scFv基因片段,构建重组噬菌粒pComb3XSS-scFv,电转化到大肠杆菌XL1-Blue构建噬菌体单链抗体库。

结果

构建鼠源性的噬菌体单链抗体文库其重组率为78%,库容量为3×108

结论

成功构建鼠源单链抗体噬菌体文库,为创伤弧菌感染的快速诊断奠定基础。

Objective

To obtain highly specific VvhA cytolysin single-chain antibody, we used phage display technology to establish a murine phage single-chain antibody library.

Methods

After immunizing BALB/c mice with cytolysin protein, the total RNA was extracted from the spleen, and cDNA was obtained by reverse transcription. The cDNA was used as a template to amplify the antibody light chain and heavy chain variable region gene fragments, and OE-PCR technology was used to splice a complete scFv gene fragment. The recombinant phagemid pComb3XSS-scFv was constructed and electrotransformed into E. coli XL1-Blue to construct a phage single-chain antibody library.

Results

The recombination rate of the mouse-derived single-chain antibody phage library was 78% and the library capacity was 3×108.

Conclusion

The murine single-chain antibody phage library is successfully constructed and lays the foundation for the early diagnosis of Vibrio vulnificus infection.

图1 小鼠免疫后血清效价分析
图2 小鼠的脾细胞抽提总RNA结果
图3 PCR扩增轻链可变区Vλ
图4 PCR扩增轻链可变区Vκ
图5 PCR扩增重链可变区VΗ
图6 OE-PCR扩增拼接scFv基因片段
图7 电转化后重组子的PCR验证
图8 图8 重组质粒pComb3XSS-scFv的酶切验证
图9 pComb3XSS-scFv噬菌体库容
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