[Prokaryotic expression, purification and antigenicity identification of human renal cell carcinoma-associated antigen G250].

Abstract

OBJECTIVE:

To amplify human renal cell carcinoma (RCC)-associated antigen G250 gene and construct a recombinant plasmid pET-42a-hG250, express and purify human G250 protein and identify its antigenicity.

METHODS:

The gene of human G250 was amplified from pGEM-T-G250 by PCR. After sequencing, the PCR product (112-1242 bp) was cloned into pET-42a prokaryotic expression vector to construct the recombinant plasmid pET-42a-hG250. The plasmid was transformed into BL21 (DE3) and human G250 protein was expressed under the induction of IPTG. The fusion protein was purified and identified by SDS-PAGE, Western blotting and ELISA sequentially.

RESULTS:

The human G250 prokaryotic expression vector pET-42a-hG250 was successfully constructed as confirmed by enzyme digestion and DNA sequencing. After transformation into BL21 (DE3), the target protein was successfully induced to express and purified as expected. Western blotting and ELISA demonstrated that the purified human G250 protein had a desirable immunogenicity.

CONCLUSION:

The recombinant prokaryotic expression vector pET-42a-hG250 has been constructed successfully. The purified human G250 protein has a good antigenicity.

• Authors: Yi Xiao, Jiangping Gao, Kun Gao, Jinqi Yan, Liang Zhang, Yu Wang, Yuanji Xu, Wei Wang, Xiaoxiong Wang, Jiyun Yu
• Journal: Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology (Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi) Vol. 29 Issue 3 Pg. 269-72 (Mar 2013) ISSN: 1007-8738 [Print] China
• PMID: 23643084 (Publication Type: Journal Article)

Chemical References

• Antigens, Neoplasm
• Recombinant Fusion Proteins
• antigen G250

Topics

• Antigens, Neoplasm (biosynthesis, genetics, immunology, isolation & purification)
• Carcinoma, Renal Cell (immunology, pathology, therapy)
• Cloning, Molecular
• Enzyme-Linked Immunosorbent Assay
• Escherichia coli (genetics)
• Gene Expression
• Genetic Engineering (methods)
• Humans
• Immunotherapy
• Kidney Neoplasms (immunology, pathology, therapy)
• Plasmids (genetics)
• Recombinant Fusion Proteins (biosynthesis, genetics, immunology, isolation & purification)