title: [Cloning of Human canstatin gene and expression of its recombinant protein].
abstract: OBJECTIVE
  To clone human canstatin gene and express its recombinant protein.
  
  
  METHODS
  The total RNA was extracted from human placenta. The canstatin gene fragment was synthesized and amplified from the total RNA by RT-PCR. The resulting product was cloned into pUCm-T vector and transformed into E.coli DH5alpha through electroporation. The gene was sequenced by the Sanger Dideoxy-mediated chain-termination method, and then the canstatin cDNA was cloned into the BamHI and HindIII sites of plasmid pET-22b (+) and transformed into E.coli BL21 where it was induced to express proteins by isopropyl-1-thio-b-Dgalactopyranoside (IPTG).
  
  
  RESULTS
  The extracted total RNA was separated into three clear bands indicating 28S, 18S, and 5S after electrophoresis. The canstatin gene fragment was synthesized and amplified from the total RNA by RT-PCR. The resulting products were cloned into pUCm-T vectors, and then were transformed into E.coli DHSa. After an over night culture, both blue and white colonies were found on the agar plate. Six white colonies were selected and cut by BamHI and HindIII. The plasmids DNA in one white colony showed one band near the location of primary plasmid after digested by BamHI and two bands near the locations of primary plasmid and objective gene fragment after digested by HindIII. The cloned gene in this white colony was sequenced and demonstrated to have the same sequence as that of canstatin gene in GenBank. Then canstatin cDNA was cut down from pUCm-T with BamHI and HindIII and ligated into the vector pET-22b (+). The resultant plasmid pET-22b (+)/canstatin was then transformed into E.coli BL21. White colonies were found on LB agar plate. Seven of them were selected and their plasmids were digested with both BamHI and HindIII. After electrophoresis, all selected colonies showed two specific bands, one was found near the location of primary plasmids, and the other near that of objective gene fragment. After IPTG induction, there was a new protein band about Mr 24 000 on SDS-PAGE. As estimated by densitometry, the percentage of the expressed product over total bacterial proteins was 18.2%, 18.8%, 23.0% and 23.4%, respectively, 1, 2, 3, and 4 hours after induction.
  
  
  CONCLUSIONS
  Human canstatin gene was successfully cloned and its recombinant proteins were expressed in this study.
authors.affiliation: Department of Gastroenterology, Changhai Hospital, the Second Military Medical University, Shanghai 200433, China. zhsli@81890.net
authors.forename: Zhao-shen
authors.initials: ZS
authors.lastname: Li
authors.forename: Xiao-ping
authors.initials: XP
authors.lastname: He
authors.forename: Zhen-xing
authors.initials: ZX
authors.lastname: Tu
authors.forename: Jun
authors.initials: J
authors.lastname: Gao
authors.forename: Xue
authors.initials: X
authors.lastname: Pan
authors.forename: Yan-fang
authors.initials: YF
authors.lastname: Gong
authors.forename: Jing
authors.initials: J
authors.lastname: Jin
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D024141:Collagen Type IV / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C502625:COL4A2 protein, human; D024141:Collagen Type IV; D010446:Peptide Fragments; D011994:Recombinant Proteins