title: [Cloning and expression of transferrin protein from Culex pipiens pallens and a study of its antimicrobial activity].
abstract: OBJECTIVE
  To clone and express transferrin (Tsf) from Culex pipiens pallens in Pichia pastoris, and detect its antibacterial activity.
  
  
  METHODS
  The coding region of transferrin from Culex pipiens pallens was amplified by RT-PCR. The product of RT-PCR was inserted into the downstream of gene encoding a-factor signal sequence in a Pichia pastoris secreting expression vector pGAPZalpha-A. The recombinant pGAPZalpha-A-Tsf vector was transformed into P. pastoris GS115 by electroporation. Recombinant strains pGAPZa-A-Tsf/GS115 were screened by Zeocin resistance and PCR. Recombinant protein was detected by SDS-PAGE and Western blotting. The recombinant transferrin protein was purified by using Ni-NTA resin. The antibacterial activity of the purified transferrin against Escherichia coli was detected.
  
  
  RESULTS
  The transferrin gene with 2,100 bp was obtained by RT-PCR. The product of recombinant plasmid pGAPZalpha-A-Tsf was approximately 2 127 bp by double digestion with restriction enzymes, consistent with the anticipated fragment length. Sequencing results showed that the inserted sequence was correct. PCR result showed that the recombinant plasmid pGAPZalpha-A-Tsf/GS115 was constructed. The results of SDS-PAGE and Western blotting showed that the relative molecular weight (Mr) of the protein was about 80,200. The recombinant transferrin protein showed antibacterial activity against Escherichia coli, and the minimum concentration was 0.25 mg/ml.
  
  
  CONCLUSIONS
  The recombinant transferrin protein from Culex pipiens pallens has been expressed in P. pastoris, and shows antibacterial activity against E. coli.
authors.forename: Song-Quan
authors.initials: SQ
authors.lastname: Wu
authors.forename: Guang-Li
authors.initials: GL
authors.lastname: Wang
authors.forename: Wu
authors.initials: W
authors.lastname: Zhou
authors.forename: Shi-Gen
authors.initials: SG
authors.lastname: Zhong
mesh_terms: D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology*; D015153:Blotting, Western; D003001:Cloning, Molecular; D003465:Culex / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects*; D015870:Gene Expression; D005822:Genetic Vectors; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins; D014168:Transferrin / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D000890:Anti-Infective Agents; D011994:Recombinant Proteins; D014168:Transferrin