盾叶半夏凝集素基因的克隆及功能分析

摘要

为克隆盾叶半夏凝集素基因，并对其功能进行分析。采用 RACE 技术从盾叶半夏中克隆得到盾叶半夏凝集素基因的全长 cDNA 序列，命名为 ppl。同时，构建了 ppl 基因的原核表达载体，并成功实现了33 kDa 重组蛋白在E．coli BL21中的表达。纯化后的重组蛋白 PPL 用于凝血和体外抗癌试验。该基因的全长 cDNA 序列为1504 bp，其中开放性阅读框（ORF）813 bp，编码270个氨基酸，具有3个甘露糖结合识别位点。PPL 具有凝血活性，这种凝血活性可受甘露糖抑制。PPL 对人鼻咽癌细胞（CNE）、人宫颈癌细胞（Hela）及人乳腺癌细胞（Bcap-37）的生长均具有抑制作用，其中对 Hela 细胞的抑制效果最好。为进一步研究 PPL 蛋白的功能奠定了理论基础。%To clone a lectin gene from Pinellia peltata and analyze its bioactivity.A lectin gene,designated as ppl,was cloned by using RACE in Pinellia peltata.The open reading frame (ORF)of P.peltata lectin (ppl)was constructed into the pET-28a vector.A recombinant protein about 33 kDa in the Escherichia coli BL21 was induced. The purified recombinant protein was used blood coagulation experiment and anticancer experiment in vitro.The full-length cDNA of ppl was 1 504 bp,and contained an 813 bp ORF with a deduced amino acid sequence of 270 res-idues.The putative PPL protein contained three mannose binding site.The PPL had clotting activity,and this activity could be inhibited by mannose.The result of anticancer experiment in vitro showed that the PPL could inhibit the growth of nasopharyngeal carcinoma cell (CNE),human cervical carcinoma cell (Hela)and human breast cancer cell (Bcap-37).These results were useful for further determination of the function of P.peltata lectin protein (PPL).