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[Cloning, ligation and expression of the variable region genes of the monoclonal antibody against human HnRNPA2/B1].
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2004 Dec; 21(6):548-51.ZY

Abstract

OBJECTIVE

To clone the variable region genes of the monoclonal antibody (McAb) against human heterogeneous nuclear ribonucleoprotein A2/B1 (HnRNPA2/B1), ligate them to assemble single chain Fv (ScFv) gene and express in Escherichia coli.

METHODS

The specificity of the anti-HnRNPA2/B1 McAb 3E8 to synthetic HnRNPA2/B1 peptide, HnRNPA2/B1 protein in lung cancer cells were examined by dot-immunobinding assay, Western blot and immunohistochemistry. The variable region genes of heavy chain (VH) and light chain (VL) were amplified from hybridoma cell by reverse transcription-polymerase chain reaction(RT-PCR), and then were linked by a linker peptide using SOE-PCR (splicing by overlap extension-PCR) to construct recombination ScFv gene. The latter was cloned into the expression vector pET28 (a+) and expressed in E coli BL21. The expressed product was identified by SDS-PAGE and competitive ELISA inhibition test.

RESULTS

It was shown that the McAb combined specifically with synthetic HnRNPA2/B1 peptide and HnRNPA2/B1 protein in three lung cancer cells. The cloned VH gene and VL gene were 345 bp and 309 bp respectively and were linked successfully to obtain ScFv gene. The ScFv protein was expressed in the form of inclusion body, with molecular weight of 28,000 and immunoreactivity to HnRNPA2/B1.

CONCLUSION

VH gene, VL gene and ScFv gene of anti-HnRNPA2/B1 antibody were cloned, constructed and functionally expressed in E coli. These results provide the experimental basis for elucidating the role of HnRNPA2/B1 in lung cancer.

Authors+Show Affiliations

Department of Immunology, School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, Sichuan, 610041 P. R. China.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

English Abstract
Journal Article

Language

chi

PubMed ID

15583979

Citation

Wang, Xia, et al. "[Cloning, Ligation and Expression of the Variable Region Genes of the Monoclonal Antibody Against Human HnRNPA2/B1]." Zhonghua Yi Xue Yi Chuan Xue Za Zhi = Zhonghua Yixue Yichuanxue Zazhi = Chinese Journal of Medical Genetics, vol. 21, no. 6, 2004, pp. 548-51.
Wang X, Peng XD, Li G, et al. [Cloning, ligation and expression of the variable region genes of the monoclonal antibody against human HnRNPA2/B1]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2004;21(6):548-51.
Wang, X., Peng, X. D., Li, G., Hu, L. J., & Bi, J. H. (2004). [Cloning, ligation and expression of the variable region genes of the monoclonal antibody against human HnRNPA2/B1]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi = Zhonghua Yixue Yichuanxue Zazhi = Chinese Journal of Medical Genetics, 21(6), 548-51.
Wang X, et al. [Cloning, Ligation and Expression of the Variable Region Genes of the Monoclonal Antibody Against Human HnRNPA2/B1]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2004;21(6):548-51. PubMed PMID: 15583979.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [Cloning, ligation and expression of the variable region genes of the monoclonal antibody against human HnRNPA2/B1]. AU - Wang,Xia, AU - Peng,Xiao-dong, AU - Li,Guang, AU - Hu,Li-juan, AU - Bi,Jian-hong, PY - 2004/12/8/pubmed PY - 2005/8/18/medline PY - 2004/12/8/entrez SP - 548 EP - 51 JF - Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics JO - Zhonghua Yi Xue Yi Chuan Xue Za Zhi VL - 21 IS - 6 N2 - OBJECTIVE: To clone the variable region genes of the monoclonal antibody (McAb) against human heterogeneous nuclear ribonucleoprotein A2/B1 (HnRNPA2/B1), ligate them to assemble single chain Fv (ScFv) gene and express in Escherichia coli. METHODS: The specificity of the anti-HnRNPA2/B1 McAb 3E8 to synthetic HnRNPA2/B1 peptide, HnRNPA2/B1 protein in lung cancer cells were examined by dot-immunobinding assay, Western blot and immunohistochemistry. The variable region genes of heavy chain (VH) and light chain (VL) were amplified from hybridoma cell by reverse transcription-polymerase chain reaction(RT-PCR), and then were linked by a linker peptide using SOE-PCR (splicing by overlap extension-PCR) to construct recombination ScFv gene. The latter was cloned into the expression vector pET28 (a+) and expressed in E coli BL21. The expressed product was identified by SDS-PAGE and competitive ELISA inhibition test. RESULTS: It was shown that the McAb combined specifically with synthetic HnRNPA2/B1 peptide and HnRNPA2/B1 protein in three lung cancer cells. The cloned VH gene and VL gene were 345 bp and 309 bp respectively and were linked successfully to obtain ScFv gene. The ScFv protein was expressed in the form of inclusion body, with molecular weight of 28,000 and immunoreactivity to HnRNPA2/B1. CONCLUSION: VH gene, VL gene and ScFv gene of anti-HnRNPA2/B1 antibody were cloned, constructed and functionally expressed in E coli. These results provide the experimental basis for elucidating the role of HnRNPA2/B1 in lung cancer. SN - 1003-9406 UR - https://www.unboundmedicine.com/medline/citation/15583979/[Cloning_ligation_and_expression_of_the_variable_region_genes_of_the_monoclonal_antibody_against_human_HnRNPA2/B1]_ L2 - https://medlineplus.gov/lungcancer.html DB - PRIME DP - Unbound Medicine ER -