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[Cloning of the variable region genes from hybridoma against bFGF and expression of single chain antibody fragments in E.coli HB2151].
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 Dec; 23(12):1150-3.XB

Abstract

AIM

To construct and express the single chain antibody (scFv) in E.coli HB2151 by cloning the variable region genes from hybridoma against bFGF.

METHODS

Total RNA was extracted from hybridoma cell line B2F3 secreting mAbs against bFGF and the cDNA was amplified by retropolymerase chain reaction (RT-PCR). V(L) and V(H) were fused by a short peptide linker containing 15 amino acids (Gly(4)Ser)(3) using splice-overlap extension PCR to construct the scFv gene. The sequences of the scFv were analyzed by Shanghai Sangon Biological Engineering Technology and Services Co. Ltd and Ig Blast data base in GenBank. The scFv gene was inserted into pCANTAB-5E vector and expressed in E.coli HB2151.

RESULTS

The V(H) gene contained 375 base pairs and encoded 125 amine acid residues. The V(L) gene contained 399 base pairs and encoded 133 amine acid residues. There were four FRs, three CDRs and two characteristic cysteine residues in the V(H) gene and the V(L) gene, respectively. The scFv gene contained 789 base pairs and encoded 263 amine acid residues with the structure of V(H)-linker-V(L). Restriction endonuclease digestion and DNA sequencing proved that the expression vector of pCANTAB-5E-scFv was constructed correctly. SDS-PAGE and ELISA analysis showed that scFv was successfully expressed in E.coli HB2151 and the expression protein had specific antigen binding activity.

CONCLUSION

The variable region genes of anti-bFGF mAbs have been cloned successfully and single chain antibody fragments have been constructed and expressed, which will be a great help to the study of humanized antibodies against bFGF.

Authors+Show Affiliations

Center of Antibody Engineering, Jinan University, Guangzhou 510632, China. anghonghlj@sohu.comNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

English Abstract
Journal Article

Language

chi

PubMed ID

18062890

Citation

Wang, Hong, et al. "[Cloning of the Variable Region Genes From Hybridoma Against bFGF and Expression of Single Chain Antibody Fragments in E.coli HB2151]." Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi = Chinese Journal of Cellular and Molecular Immunology, vol. 23, no. 12, 2007, pp. 1150-3.
Wang H, Chen D, Deng N, et al. [Cloning of the variable region genes from hybridoma against bFGF and expression of single chain antibody fragments in E.coli HB2151]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007;23(12):1150-3.
Wang, H., Chen, D., Deng, N., Xiang, J. J., Jin, Y. J., Huang, H. L., Tang, Y., & Yang, H. Y. (2007). [Cloning of the variable region genes from hybridoma against bFGF and expression of single chain antibody fragments in E.coli HB2151]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi = Chinese Journal of Cellular and Molecular Immunology, 23(12), 1150-3.
Wang H, et al. [Cloning of the Variable Region Genes From Hybridoma Against bFGF and Expression of Single Chain Antibody Fragments in E.coli HB2151]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007;23(12):1150-3. PubMed PMID: 18062890.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [Cloning of the variable region genes from hybridoma against bFGF and expression of single chain antibody fragments in E.coli HB2151]. AU - Wang,Hong, AU - Chen,Dan, AU - Deng,Ning, AU - Xiang,Jun-Jian, AU - Jin,Ying-Jie, AU - Huang,Hong-Liang, AU - Tang,Yong, AU - Yang,Hong-Yu, PY - 2007/12/8/pubmed PY - 2009/5/7/medline PY - 2007/12/8/entrez SP - 1150 EP - 3 JF - Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology JO - Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi VL - 23 IS - 12 N2 - AIM: To construct and express the single chain antibody (scFv) in E.coli HB2151 by cloning the variable region genes from hybridoma against bFGF. METHODS: Total RNA was extracted from hybridoma cell line B2F3 secreting mAbs against bFGF and the cDNA was amplified by retropolymerase chain reaction (RT-PCR). V(L) and V(H) were fused by a short peptide linker containing 15 amino acids (Gly(4)Ser)(3) using splice-overlap extension PCR to construct the scFv gene. The sequences of the scFv were analyzed by Shanghai Sangon Biological Engineering Technology and Services Co. Ltd and Ig Blast data base in GenBank. The scFv gene was inserted into pCANTAB-5E vector and expressed in E.coli HB2151. RESULTS: The V(H) gene contained 375 base pairs and encoded 125 amine acid residues. The V(L) gene contained 399 base pairs and encoded 133 amine acid residues. There were four FRs, three CDRs and two characteristic cysteine residues in the V(H) gene and the V(L) gene, respectively. The scFv gene contained 789 base pairs and encoded 263 amine acid residues with the structure of V(H)-linker-V(L). Restriction endonuclease digestion and DNA sequencing proved that the expression vector of pCANTAB-5E-scFv was constructed correctly. SDS-PAGE and ELISA analysis showed that scFv was successfully expressed in E.coli HB2151 and the expression protein had specific antigen binding activity. CONCLUSION: The variable region genes of anti-bFGF mAbs have been cloned successfully and single chain antibody fragments have been constructed and expressed, which will be a great help to the study of humanized antibodies against bFGF. SN - 1007-8738 UR - https://www.unboundmedicine.com/medline/citation/18062890/[Cloning_of_the_variable_region_genes_from_hybridoma_against_bFGF_and_expression_of_single_chain_antibody_fragments_in_E_coli_HB2151]_ L2 - https://www.lens.org/lens/search?q=citation_id:18062890 DB - PRIME DP - Unbound Medicine ER -