Please wait a minute...

中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2018, Vol. 38 Issue (11): 25-31    DOI: 10.13523/j.cb.20181104
    
Construction and Screening of a Phage Display Library of Single Chain Fv Antibody Efficiently from Mouse Immunized with Ovalbumin
Qiao-li LANG,Lin YU,Qi-lin HE,Liang-peng GE(),Xi YANG()
Chongqing Academy of Animal Sciences, Key Laboratory of Pig Industry Sciences, Chongqing Research Center for the Development and Utilization of Medical Animal Resources, Chongqing 402460, China
Download: HTML   PDF(634KB) HTML
Export: BibTeX | EndNote (RIS)      

Abstract  

Objective:to establish an efficient method for construction of a phage display library of single chain Fv (scFv) antibody efficiently from mouse immunized with ovalbumin (OVA) and obtain anti-OVA single chain antibody.Methods:Balb/C mice were immunized with OVA. The heavy chain and light chain variable region gene of immunoglobulin were amplified from mRNA of spleen cells of mice with high serum OVA antibody titer by RT-PCR and joined by a DNA linker with seamless cloning. These fragments were inserted into phage expression vector to construct a phage display library. After measurement of library capacity, affinity selection and ELISA analysis were run to find high affinity scFv. Its protein was expressed by Epxi-CHO cells and identified by Western blot analysis.Results:The OVA scFv phage display library was constructed and its library capacity was 1.2 x 10 7 cfu. Eight high affinity scFv were screened from this library. The 2# clone with the highest affinity was cloned into the eukaryotic expression vector and expressed in expi-CHO cells. The Western blot analysis showed that it is a soluble antibody. Conclusion:A highly efficient method for construction of a scFv phage library and generated a high affinity OVA scFv antibody are established. These laid a good foundation for the development of the research of OVA ELISA analysis kit.

Key words： Ovalbumin (OVA)      Single chain Fv (scFv) antibody      Phage display      Eukaryotic expression     
Received: 15 July 2018      Published: 06 December 2018
ZTFLH:  Q816  
Corresponding Authors: Liang-peng GE,Xi YANG     E-mail: geliangpeng1982@163.com;406162197@qq.com
Service
E-mail this article
Add to my bookshelf
Add to citation manager
E-mail Alert
RSS
Articles by authors
Qiao-li LANG
Lin YU
Qi-lin HE
Liang-peng GE
Xi YANG
Cite this article:

Qiao-li LANG,Lin YU,Qi-lin HE,Liang-peng GE,Xi YANG. Construction and Screening of a Phage Display Library of Single Chain Fv Antibody Efficiently from Mouse Immunized with Ovalbumin. China Biotechnology, 2018, 38(11): 25-31.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20181104     OR     https://manu60.magtech.com.cn/biotech/Y2018/V38/I11/25

Name Sequence
VH-For GGCCAGCCGGCC1)ATGGCCCAGGTSMARCTGCAGSAGTCWGG
VH-Rev TCCACCTGAGGAGA2)CGGTGACCGTGGTCCCTTGGCCCC
Linker GTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATCGAGCTC
Vκ-For TCGGACATCGAGCTCGACATTGAGCTCACCCAGTCTCCA
Vκ1- Rev GCGGCGCCCGTTTGATTTCCAGCTTGGTGCC
Vκ2- Rev GCGGCGCCCGTTTTATTTCCAGCTTGGTCCC
Vκ4- Rev GCGGCGCCCGTTTTATTTCCAACTTTGTCCC
Vκ5 - Rev GCGGCGCCCGTTTCAGCTCCAGCTTGGTCCC
Table1 List of primers
Fig.1 Construction of scFv phagemid vector
Fig.2 PCR amplification of VH and Vκ geneM:DL2000 maker; 1:PCR product of VH; 2:PCR product of Vκ
Fig.3 PCR amplification of scFv gene M:DL2 000 maker; 1:PCR product of scFv
Fig.4 Identification of individual recombinant clones by PCR M:DL5000 maker; 1-15: Individual recombinant clones
Fig.5 ELISA selection of phage antibodies specific
Rounds of panning Input phage(cfu) Output phage(cfu) Recovery rating
1 3×1012 1.2×106 4×10-7
2 2.5×1012 3.5×105 1.4×10-7
3 0.7×1012 1.2×105 1.7×10-7
4 10×1012 7.5×106 7.5×10-7
Table 2 Enrichment of phage antibody library by panning
Fig.6 Western blot 1:Transfection of cell supernatant;
2:Unloaded somatic supernatant
[1]   Nieri P, Donadio E, Rossi S , et al. Antibodies for therapeutic uses and the evolution of biotechniques. Current Medicinal Chemistry, 2009,16(6):753-779.
doi: 10.2174/092986709787458380 pmid: 19199935
[2]   马颖, 邹全明 . 单链抗体及其在生物医学中的应用. 免疫学杂志, 2006,22(s1):1-5.
[2]   Ma Y, Zou Q M . Single chain antibody and its application in biomedicine. Immunological Journal, 2006,22(s1):1-5.
[3]   Pande J, Szewczyk M M, Grover A K . Phage display: concept, innovations, applications and future. Biotechnology Advances, 2010,28(6):849-858.
doi: 10.1016/j.biotechadv.2010.07.004 pmid: 20659548
[4]   Wu C H, Liu I J, Lu R M , et al. Advancement and applications of peptide phage display technology in biomedical science. Journal of Biomedical Science, 2016,23(1):1-14.
doi: 10.1186/s12929-015-0217-0 pmid: 4710007
[5]   Dudak F C, Boyaci I H, Orner B P . The discovery of small-molecule mimicking peptides through phage display. Molecules, 2011,16(1):774-789.
doi: 10.3390/molecules16010774 pmid: 21248663
[6]   Chan C E, Lim A P, Macary P A , et al. The role of phage display in therapeutic antibody discovery. International Immunology, 2014,26(12):649-657.
doi: 10.1093/intimm/dxu082 pmid: 25135889
[7]   佟平, 高金燕, 陈红兵 . 鸡蛋清中主要过敏原的研究进展. 食品科学, 2007,28(8):565-568.
doi: 10.3321/j.issn:1002-6630.2007.08.143
[7]   Tong P, Gao J Y, Chen H B . Research progress of major egg white allergens. Food Science, 2007,28(8):565-568.
doi: 10.3321/j.issn:1002-6630.2007.08.143
[8]   Huntington J A, Stein P E . Structure and properties of ovalbumin. Journal of Chromatography B Biomedical Sciences & Applications, 2001,756(1-2):189-198.
doi: 10.1016/S0378-4347(01)00108-6 pmid: 11419711
[9]   Thie H, Meyer T, Schirrmann T , et al. Phage display derived therapeutic antibodies. Current Pharmaceutical Biotechnology, 2008,9(6):439-446.
doi: 10.2174/138920108786786349
[10]   Hoogenboom H R . Selecting and screening recombinant antibody libraries. Nature Biotechnology, 2005,23(9):1105-1116.
doi: 10.1038/nbt1126 pmid: 16151404
[11]   Löfblom J . Bacterial display in combinatorial protein engineering. Biotechnology Journal, 2011,6(9):1115-1129.
doi: 10.1002/biot.201100129 pmid: 21786423
[12]   Bellofiore P, Petronzelli F, Martino T D , et al. Identification and refinement of a peptide affinity ligand with unique specificity for a monoclonal anti-tenascin-C antibody by screening of a phage display library. Journal of Chromatography A, 2006,1107(1-2):182-191.
doi: 10.1016/j.chroma.2005.12.064 pmid: 16414054
[13]   Jefferson T, Smith S, Demicheli V , et al. Assessment of the efficacy and effectiveness of influenza vaccines in healthy children:abstract of systematic review. Chinese Journal of Evidence-Based Medicine, 2005,365(9461):773-780.
doi: 10.1016/S0140-6736(05)17984-7 pmid: 15733718
[1] CHENG Xu,YANG Yu-qing,WU Sai-nan,HOU Qin-long,LI Yong-mei,HAN Hui-ming. Construction of DNA Vaccines of Staphylococcus aureus SarA, IcaA and Their Fusion Genes and Preliminary Study in Mouse Immune Response[J]. China Biotechnology, 2020, 40(7): 41-50.
[2] YANG Li,SHI Xiao-yu,LI Wen-lei,LI Jian,XU Han-mei. Optimization of Electroporation Conditions in Construction of Phage Display Antibody Library[J]. China Biotechnology, 2020, 40(4): 42-48.
[3] CHEN Xiu-xiu,WU Cheng-lin,ZHOU Li-jun. Research Progress in Preparation and Clinical Application of Therapeutic Human Antibodies[J]. China Biotechnology, 2019, 39(10): 90-96.
[4] Jin-jing LI,Fei XU,Yan-wei JI,Mei SHU,Zhui TU,Jin-heng FU. Biopanning of Anti c-Myc-tag Nanobodies and Its Application for Bioimaging[J]. China Biotechnology, 2018, 38(2): 61-67.
[5] PANG Qian,CHEN Jing,WANG Xiao-hong,WANG Jia. Screening of Anti-Aflatoxin B1 ScFv Based on Phage Display Technology and Analysis of Its Protein Structure[J]. China Biotechnology, 2018, 38(12): 41-48.
[6] FANG Yuan,XU Guang-xian,WANG Xian,WANG Hong-xia,PAN Jun-fei. Construction of Camelid Natural Nanobody Phage Display Library and Screening for Anti-GDH Nanobody[J]. China Biotechnology, 2018, 38(12): 49-56.
[7] HU Chang-wu, XIE Jun, ZHU Nai-shuo. Comparison of the Inhibitory Efficiency of M13 Based 7-mer and 12-mer Phage Display Libraries Derived Peptides as Tumor Necrosis Factor Alpha Antagonist[J]. China Biotechnology, 2017, 37(5): 1-8.
[8] WANG Qing, XU Yan-zhao, WEI Xiao-xiao, WANG Qiu-xia, HANG Bo-lin, SUN Ya-wei, WANG Fei-fei, HU Jian-he . Preparation of Polyclonal Antiserums of GP5a Protein of Porcine Reproductive and Respiratory Syndrome Virus[J]. China Biotechnology, 2015, 35(8): 38-43.
[9] LI You-jian, ZHANG guo-qi, Gou ji-xing, CHEN xin-kai, DOU Xiao-xia, CHEN Chuang-fu, SHENG Jin-liang. Expression of Ovine Myostatin Gene and Construction and Identification of Nanobody Library Against Recombinant MSTN[J]. China Biotechnology, 2014, 34(9): 87-93.
[10] WANG Jin-sheng, JIANG Hao-wu, ZHANG Jin-xia, PAN Lei, ZHAO Feng-zhi, YU Yun-fei, CAI Ya-xiong, DENG Ning. Optimized Expression of a Mouse-human Chimeric Antibody Production in HEK 293T Cells Against Human FGF2[J]. China Biotechnology, 2014, 34(5): 14-22.
[11] PANG Min, WANG Hai-long, GUO Min, GUO Rui. Construction of an Eukaryocyte Expression Vector of Human ANKRD49 and the Study of Function and RNA Interference Target of ANKRD49[J]. China Biotechnology, 2014, 34(10): 15-21.
[12] YUAN Li, DAI He-ping. Overview of Scaffold Protein Used for Selection of Artificial Binding Proteins[J]. China Biotechnology, 2013, 33(1): 95-103.
[13] LIU Yu-fen, DONG Li-li, SUN Yu-gang, LIU Peng, CHEN Hui, ZHAO Wen-ge. Expression and Function of Fibrinolytic Enzyme Gene from Gloydius intermediu Venom Gland[J]. China Biotechnology, 2012, 32(10): 1-6.
[14] XU Wen-qi, CHAI Xiao-jie, ZHANG Ting, DAI Jing-yu, ZHANG Xiao-lin. Construction of Trypsin Inhibitor KSTI3 Gene New Eukaryotic Expression System and Expression in Dunaliella salina[J]. China Biotechnology, 2011, 31(8): 29-34.
[15] WANG Zi-ye, FENG Juan, MA Xue-mei, WANG Ming-lian, ZHONG Ru-gang. Screening of HIV-1 p24 Binding Peptides from Phage Display Peptide Library[J]. China Biotechnology, 2011, 31(5): 104-107.
主管：中国科学院 　主办：中国科学院文献情报中心 　中国生物技术发展中心 　中国生物工程学会