title: [Cloning full-length homologous cDNAs of pollen allergens in Humulus Scandens(Lour.) Merr by degenerate primer].
abstract: OBJECTIVE
  To establish a stable and reliable method for fast cloning homologous genes of pollen allergens in allergen-containing plants.
  
  
  METHODS
  Degenerate primers were designed based on the bioinformatic analysis of numerous allergens available from the database. Subsequent amplification of the allergen genes was conducted in the weed pollen cDNA pool by a selective PCR profile. Following the truncated gene cloning, RACE method was used to isolate full-length cDNA. Gene function was deduced by sequence alignment in GenBank database. The degenerate ability of the primer was compared with the full-length cDNA sequences.
  
  
  RESULTS
  Three full-length cDNAs were obtained. Sequence analysis showed that these new genes shared as high as 79%-85% homology with a large amount of known allergen profilins and were hence regarded as members of panallergen profilin family. Comparing these genes with the degenerate primers that were initially used in truncated gene cloning revealed that alternative nucleotide degeneracy occurred beyond the degenerate site predesigned, suggesting that further degeneracy was expanded by Touchdown-gradient PCR.
  
  
  CONCLUSIONS
  Cloning of homologous genes or allergen genes can be efficiently achieved by using the combination of degenerate primer with Touchdown-gradient RT-PCR in the species such as Humulus scandens that has not yet been investigated.
authors.affiliation: Allergy and Inflammation Research Institute, Shantou University Medical College, Shantou 515031, China.
authors.forename: Ai-lin
authors.initials: AL
authors.lastname: Tao
authors.forename: Shao-heng
authors.initials: SH
authors.lastname: He
authors.forename: Li-da
authors.initials: LD
authors.lastname: Zhang
authors.forename: Zhang-quan
authors.initials: ZQ
authors.lastname: Chen
authors.forename: Dong-dong
authors.initials: DD
authors.lastname: Li
mesh_terms: D000485:Allergens / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000737:chemistry; D027582:Humulus / Q000276:immunology*; D008969:Molecular Sequence Data; D010802:Phylogeny; D011058:Pollen / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000485:Allergens; D017931:DNA Primers; D018076:DNA, Complementary