title: [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.
  
  
  CONCLUSIONS
  The recombinant prokaryotic expression vector pET-42a-hG250 has been constructed successfully. The purified human G250 protein has a good antigenicity.
authors: Department of Urology, Chinese PLA General Hospital, Beijing, China. xiaoyi1007@gmail.com
authors: Yi
authors: Y
authors: Xiao
authors: Jiangping
authors: J
authors: Gao
authors: Kun
authors: K
authors: Gao
authors: Jinqi
authors: J
authors: Yan
authors: Liang
authors: L
authors: Zhang
authors: Yu
authors: Y
authors: Wang
authors: Yuanji
authors: Y
authors: Xu
authors: Wei
authors: W
authors: Wang
authors: Xiaoxiong
authors: X
authors: Wang
authors: Jiyun
authors: J
authors: Yu
mesh_terms: D000951:Antigens, Neoplasm / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D002292:Carcinoma, Renal Cell / Q000276:immunology / Q000473:pathology* / Q000628:therapy; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D007167:Immunotherapy; D007680:Kidney Neoplasms / Q000276:immunology / Q000473:pathology* / Q000628:therapy; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000951:Antigens, Neoplasm; D011993:Recombinant Fusion Proteins; C400566:antigen G250