title: [Prokaryotic expression of recombinant prochymosin gene and its antiserum preparation].
abstract: OBJECTIVE
  To optimize the prochymosin (pCHY) gene codons and express the gene in Escherichia coli (E.coli),  and to prepare its antiserum and detect chymosin protein specifically.
  
  
  METHODS
  According to codon usage bias of  E.coli,  prochymosin gene sequence was synthesized based on the conserved sequences of prochymosin gene from bovine,  lamb and camel,  and then cloned into the  plasmid pET-30a and pcDNA3-AAT-COMP-C3d3 (pcD-ACC),  respectively. pET-30a-pCHY was expressed,  as the detected antigen,  in E.coli BL21(DE3) after IPTG induction. RT-PCR was used to detect prochymosin mRNA expression in liver from the mice injected pcDNA3-AAT-COMP-pCHY-C3d3(pACCC) by hydrodynamics-based transfection method. To prepare the antiserum of prochymosin,  pACCC and GST-pCHY proteins were used to immunize New Zealand rabbits in accordance with DNA prime-protein boost strategy. Antibody levels were tested by ELISA.
  
  
  RESULTS
  Western blotting showed the molecular weight of His-pCHY protein was about 55 000,  similar to the expected molecular size. ELISA demonstrated that the titer level of prochymosin antiserum was high.
  
  
  CONCLUSIONS
  Based on the codon optimization,  we have obtained high-titer prochymosin antiserum through DNA vaccine vector pcD-ACC combined with DNA prime-protein boost strategy,  similar to that by protein vaccine.
authors: Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China. lxyxp@sina.cn
authors: Xin-ping
authors: XP
authors: Li
authors: Huan-huan
authors: HH
authors: Liu
authors: Yan
authors: Y
authors: Pu
authors: Fu-chun
authors: FC
authors: Zhang
authors: Yi-jie
authors: YJ
authors: Li
mesh_terms: D000818:Animals; D000918:Antibody Specificity / Q000276:immunology; D012085:Chymosin / Q000235:genetics* / Q000276:immunology*; D004792:Enzyme Precursors / Q000235:genetics* / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007106:Immune Sera / Q000276:immunology* / Q000302:isolation & purification; D008099:Liver / Q000378:metabolism; D051379:Mice; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D014162:Transfection
chemical_list: D004792:Enzyme Precursors; D007106:Immune Sera; D011993:Recombinant Fusion Proteins; C032443:prorennin; D012085:Chymosin