Gene Cloning and Evaluation Activity of Recombinant Lectin Isolated from Agrocybe aegerita

• Main Authors: Jian-Nan Chen, 陳建男
• Other Authors: 張鴻民
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/57703820103214536335

碩士 === 國立臺灣大學 === 食品科技研究所 === 93 === Abstract Agrocybe aegerita (AA), one of popular duetary mushrooms in Taiwan, is well known for its good nutritional values and low calory content. Lectin of AA induced apoptosis, influenced DNase activity, and showed strong inhibition on growth of human tumour cell lines. In present study, primers were designed on the basis of the nucleotide sequence of AA lectin listed in NCBI to carry out Northern blot hybridization analysis and found that the mRNA of AA lectin was about 0.9 kb. In addition, the start site of cDNA of AA lectin was determined by 5’ RACE and found that retroposons of transcription start site was at 39 bp upstream to ATG and the full of cDNA was 672 bp, in which length of open reading frame was 489 bp. Then, nucleotide and deduced amino acid sequences of open reading frame of AA lectin were aligned by a program of CLUSTAL_X. Based on the differences of nucleotide and deduced amino acid sequences in the present sample from those listed in NCBI, we presumed that there were at least two isotypic forms of lectins in AA cultivation. For the production of recombinant protein of AA lectin, primers were designed to construct pET-28b expression vectored by nucleotide sequencing of result of 5’RACE, and then transformed into E. coli. The expression of recombinant protein by E. coli increased with the increasing incubation time for up to 3 hr, and the obtained recombinant protein was found mainly water-soluble. Hemagglutination assay revealed that recombinant protein expressed in E. coli induced agglutination with 2 % Pronase-treated rabbit RBC, while no effect was observed with normal rabbit RBC.