中文摘要：

目的用含防御素HNP-3成熟肽cDNA片段构建酵母双杂交诱饵质粒，进行自激活和毒性检测。方法培养HL-60细胞，提取RNA，RT-PCR扩增含防御素HNP-3成熟肽的cDNA片段，克隆入pBlue-script-SK-Ⅱ质粒，经测序鉴定后，再亚克隆到人酵母双杂交诱饵载体pGBKT7中，PCR及测序鉴定。重组诱饵质粒用缺陷培养基筛选方法进行自激活和毒性检测。结果构建的重组质粒pBluescript-SK-Ⅱ-HNP-3，经测序鉴定，插入片段为HNP-3成熟肽的cDNA序列。亚克隆构建的重组诱饵质pGBKT7-HNP-3，测序鉴定表明其插入目的基因序列无误。重组诱饵质粒自激活和毒性检测显示无自激活现象发生，对酵母细胞AH109无毒性作用。结论成功构建了防御素HNP-3成熟肽的酵母双杂交诱饵载体；为用酵母双杂交技术研究与防御素HNP-3成熟肽相互作用蛋白质打下了基础。

英文摘要：

Objective To construct the bait plasmid of HNP-3 mature peptide in yeast two-hybrid system and examine whether the recombinant bait plasmid has self-activating and toxicity effect. Methods Using RT-PCR technique, the cDNA fragments of HNP-3 mature peptide gene were amplified from the extracted RNA in cultured HL-60 cells. The fragment was firstly cloned into pBluescript-SK-Ⅱ vector, confirmed by sequencing,then sub-cloned into the bait plasmid pGBKT7 and identified with PCR and sequence analysis techniques. The recombinant plasmid was introduced into the yeast cell AH109,and its self-activating and toxicity effect was tested by auxotrophic selective culture. Results DNA sequencing indicated that the inserted fragment in pBluescript-SK-Ⅱ vector was HNP-3 mature peptide gene sequence, and the sub-cloned recombinant pGBKT7-HNP-3 was no mismatch. The recombinant bait plasmid didn＇ t have self-activating effect and did not show toxicity to yeast AH109 cell. Conclusion The bait plasmid of HNP-3 mature peptide was constructed successfully. This was helpful for investigating the proteins interacting with HNP-3 mature peptide by yeast two-hybrid technique.