Preparation and inentification of camel nanobodies against human G250 ectodomain

Ming Hu; Luofu Wang; Jun Jiang; Yanli Guo; Xiaozhou Fan; Zhouquan Li; Zhiping Yu; Tong Zhu

doi:10.16016/j.1000-5404.201611198

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Preparation and inentification of camel nanobodies against human G250 ectodomain

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- Preparation and inentification of camel nanobodies against human G250 ectodomain
- Journal of Third Military Medical University Vol. 39, Issue 6, Pages: 541-547(2017)
- 作者机构：
  
  1.400042 重庆，第三军医大学大坪医院野战外科研究所泌尿外科
  2.400038 重庆，第三军医大学西南医院超声科
  3.430072 武汉，中国科学院水生生物研究所淡水生态与生物技术国家重点实验室
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(81472399)
- DOI：10.16016/j.1000-5404.201611198
  CLC： R392-33;R32.11
- Received：23 November 2016，
  
  Revised：2016-12-20，
  
  Published：2017-06
- 稿件说明：
移动端阅览
Ming Hu, Luofu Wang, Jun Jiang, et al. Preparation and inentification of camel nanobodies against human G250 ectodomain[J]. Journal of Third Military Medical University, 2017, 39(6): 541-547.
DOI：

Ming Hu, Luofu Wang, Jun Jiang, et al. Preparation and inentification of camel nanobodies against human G250 ectodomain[J]. Journal of Third Military Medical University, 2017, 39(6): 541-547. DOI： 10.16016/j.1000-5404.201611198.

摘要

目的

制备能与肾癌相关抗原G250胞外区特异性结合的纳米抗体。

方法

通过瞬时转染方式将包含G250蛋白胞外区基因片段的质粒导入到真核细胞HEK 293F中表达G250胞外区，以该重组蛋白免疫新疆双峰驼，构建噬菌体展示纳米抗体库。通过体外定向筛选，得到能与G250蛋白胞外区特异性结合的噬菌体，以Monoclonal phage ELISA方法筛选出具有结合活性的噬菌体克隆。通过细胞ELISA和流式细胞仪再次验证淘选到的纳米抗体的细胞结合活性。

结果

测序证明编码G250的重组DNA片段序列正确，Western blot检测结果证实G250重组蛋白成功表达。通过免疫新疆双峰驼，成功构建了库容为2.87×10

的噬菌体展示骆驼VHH免疫文库。在体外对免疫纳米抗体库进行3轮固相筛选，使具有结合活性的噬菌体克隆得到了有效富集，阳性率高达54.3%。经过抗原ELISA、细胞ELISA鉴定，筛选出3个具有细胞结合活性的纳米抗体噬菌体克隆，取其中结合活性最高的Nb-G5进行表达、纯化，产量为1.5 mg/L培养物，流式细胞仪检测结果证实Nb-G5与G250表达阳性的细胞具有很强的特异性结合能力。

结论

从纳米抗体库中淘选得到了在分子和细胞水平均能与G250蛋白胞外区特异性结合的纳米抗体。

Abstract

Objective

To prepare nanobodies capable of specifically binding to human G250 ectodomain and provide a basis for diagnostic and therapeutic application of renal cell carcinoma.

Methods

The plasmid harboring the DNA fragment of human G250 ectodomain was transiently transfected into HEK 293F cells for protein expression. Two Xinjiang Bactrian camels were immunized with the recombinant G250 ectodomain protein. VHH fragments were isolated by nested PCR from lymphocyte cDNA. A VHH nanobody library was constructed using phage display technology. Nanobodies specifically recognizing G250 were enriched by biopanning

in vitro

and identified by monoclonal phage ELISA. Cell binding ability of the selected nanobodies was further characterized by cell ELISA and flow cytometry.

Results

The recombinant DNA fragment encoding human G250 ectodomain was confirmed by DNA sequencing

and the recombinant G250 protein was proved to be successfully expressed by Western blotting. After immunizing the 2 Xinjiang Bactrian camels with the recombinant G250 protein

the phage display VHH library containing 2.87×10

in size was constructed. After 3 rounds of biopanning against the G250 protein

in vitro

the G250-specific VHH clones were efficiently enriched with a positive rate of 54.3%.A clone Nb-G5 with the highest cell binding activity was identified upon 3 VHH clones selected from the phage display VHH library. Nb-G5 was expressed in a soluble form with a yield of 1.5 mg/L cell culture

and it presented high selectivity and specificity to the cells expressing G250 protein.

Conclusion

Nanobodies

which specifically bind to G250 ectodomain in molecular and cellular levels

are successfully prepared.

关键词

Keywords

references

Oosterwijk E, Ruiter D J, Hoedemaeker P J, et al. Monoclonal antibody G 250 recognizes a determinant present in renal-cell carcinoma and absent from normal kidney[J]. Int J Cancer, 1986, 38(4): 489-494. DOI:10.1002/ijc.2910380406

[Grabmaier K, Vissers J, De Weijert M, et al. Molecular cloning and immunogenicity of renal cell carcinoma-associated antigen G250[J]. Int J Cancer, 2000, 85(6): 865-870. DOI:10.1002/(sici)1097-0215(20000315)85:6<865::aid-ijc21>3.0.co;2-q

阳孟君, 王洛夫, 郭燕丽, 等.携载G250单克隆抗体的纳米微泡靶向于肾细胞癌的体内外实验研究[J].第三军医大学学报, 2016, 38(2): 112-118. DOI:10.16016/j.1000-5404.201508106

Yang M J, Wang L F, Guo Y L, et al. In vivo and in vitro study of nanoscale microbubbles with G250 monoclonal antibodies targeting to renal cell carcinoma[J]. J Third Mil Med Univ, 2016, 38(2): 112-118. DOI:10.16016/j.1000-5404.201508106

徐沐琳, 张永学. Nanobody在分子影像诊断和分子靶向治疗研究中的应用进展[J].华中科技大学学报:医学版, 2014, 43(4): 486-488. DOI:10.3870/j.issn.1672-0741.2014.04.029

Xu M L, Zhang Y X. Application of Nanobody in molecular imaging diagnosis and molecular targeted therapy[J]. Acta Medicinae Universitatis Scientiae et Technologiae Huazhong, 2014, 43(4): 486-488. DOI:10.3870/j.issn.1672-0741.2014.04.029

Conrath K E, Lauwereys M, Galleni M, et al. Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae[J]. Antimicrob Agents Chemother, 2001, 45(10): 2807-2812. DOI: 10.1128/AAC.45.10.2807-2812.2001

Vincke C, Gutiérrez C, Wernery U, et al. Generation of single domain antibody fragments derived from camelids and generation of manifold constructs[J]. Methods Mol Biol, 2012, 907: 145-176. DOI:10.1007/978-1-61779-974-7_8

Hamers-Casterman C, Atarhouch T, Muyldermans S, et al. Naturally occurring antibodies devoid of light chains[J]. Nature, 1993, 363(6428): 446-448. DOI: 10.1038/363446a0

Nguyen V K, Hamers R, Wyns L, et al. Loss of splice consensus signal is responsible for the removal of the entire C (H)1 domain of the functional camel IGG2A heavy-chain antibodies[J]. Mol Immunol, 1999, 36(8): 515-524.

Muyldermans S, Baral T N, Retamozzo V C, et al. Camelid immunoglobulins and nanobody technology[J]. Vet Immunol Immunopathol, 2009, 128(1/3): 178-183. DOI:10.1016/j.vetimm.2008.10.299

郭婷, 张宇昊, 马良.纳米抗体的特性及其应用研究进展[J].食品科学, 2013, 34(3): 294-297.

Guo T, Zhang Y H, Ma L. Properties and application prospects of nano-antibody in food industry[J]. Food Science, 2013, 34(3): 294-297.

Bos R, Zhong H, Hanrahan C F, et al. Levels of hypoxia-inducible factor-1 alpha during breast carcinogenesis[J]. J Natl Cancer Inst, 2001, 93(4): 309-314. DOI:10.1093/jnci/93.4.309

Bos R, van der Groep P, Greijer A E, et al. Levels of hypoxia-inducible factor-1alpha independently predict prognosis in patients with lymph node negative breast carcinoma[J]. Cancer, 2003, 97(6): 1573-1581. DOI:10.1002/cncr.11246

McKiernan J M, Buttyan R, Bander N H, et al. Expression of the tumor-associated gene MN: a potential biomarker for human renal cell carcinoma[J]. Cancer Res, 1997, 57(12): 2362-2365.

Bleumer I, Knuth A, Oosterwijk E, et al. A phase Ⅱ trial of chimeric monoclonal antibody G250 for advanced renal cell carcinoma patients[J]. Br J Cancer, 2004, 90(5): 985-990.DOI:10.1038/sj.bjc.6601617

Tu Z, Xu Y, He Q, et al. Isolation and characterisation of deoxynivalenol affinity binders from a phage display library based on single-domain camelid heavy chain antibodies (VHHs)[J]. Food and Agricultural Immunology, 2012, 23(2): 123-131. DOI:10.1080/09540105.2011.606560

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