title: [Prokaryotic expression, purification of HSP65-MUC1 VNTR2 fusion protein and primary research on its tumoricidal effect].
abstract: OBJECTIVE
  To express the HSP65-MUC1 VNTR(2) in E.coli and to evaluate its activity of inhibiting tumor growth in vivo.
  
  
  METHODS
  HSP65 and MUC1 VNTR(2) were generated by PCR method and sub-cloned to pET28a(+) to construct the recombinant expression vector HSP65-MUC1 VNTR(2)-pET28a(+). E.coli BL21(DE3) bearing the plasmid was induced with IPTG for protein production. Target protein was characterized by Western blot with monoclonal antibody and purified by Q-Sepharose ion-exchange chromatography and gel filtration. The murine cancer cell linejB16 that transfected by human gene MUC1 was utilized to construct the model of carcinoma, and the tumor growth inhibition activities of HSP65-MUC1VNTR(2) was evaluated in mice C57BL/6.
  
  
  RESULTS
  The gene HSP65 and MUC1 VNTR(2) confirmed by sequence analysis matched respectively with BCG HSP65 and human gene MUC1 VNTRs in GenBank exactly. The reconstructed vector HSP65-MUC1 VNTR(2)-pET28a could express target protein stably in the soluble fraction of bacterial extract. The purity of HSP65-MUC1 VNTR(2) protein could be above 95% after purification by Q ion-exchange chromatography and gel filtration. The result of Western blot with monoclonal antibody showed positive. The results of prophylactic immunization with HSP65-MUC1 VNTR(2) fusion protein showed that experiment all groups had significantly higher tumor inhibition rates than that of control group.
  
  
  CONCLUSIONS
  In summary, HSP65-MUC1 VNTR(2) fusion protein was solubly expressed in prokaryotic expression system and its tumor growth inhibition activity was evaluated primarily. The result indicated that the fusion protein could inhibit the MUC1 positive tumor growth significantly. It can be used in the future research as the cancer vaccine.
authors.affiliation: Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Yu-Ling
authors.initials: YL
authors.lastname: Zheng
authors.forename: Hua
authors.initials: H
authors.lastname: Jiang
authors.forename: Xi-Liang
authors.initials: XL
authors.lastname: Wang
authors.forename: Yong-Qiang
authors.initials: YQ
authors.lastname: Jiang
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000302:isolation & purification / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D018834:Chaperonin 60; D018833:Chaperonins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D051379:Mice; D018598:Minisatellite Repeats / Q000235:genetics*; D018396:Mucin-1 / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D009163:Mycobacterium bovis; D011387:Prokaryotic Cells / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D001426:Bacterial Proteins; D018834:Chaperonin 60; C060192:MUC1 protein, human; D018396:Mucin-1; D011993:Recombinant Fusion Proteins; C074119:heat-shock protein 65, Mycobacterium; D018833:Chaperonins