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荷斯坦奶牛杀菌/通透性增加蛋白N端cDNA在大肠杆菌中的融合表达
  • 期刊名称:农业生物技术学报.
  • 时间:0
  • 页码:599-602
  • 语言:中文
  • 分类:S188[农业科学—农业基础科学]
  • 作者机构:[1]安徽农业大学动物科技学院,合肥230036
  • 相关基金:国家高技术研究与发展计划(863)项目(No.2006AA10Z320)、国家自然科学基金项目(No.30871851、No.30800811)和安徽省教育厅自然科学研究项目(No.KJ2007A021)资助.
  • 相关项目:抗ACTH肽对鸡应激性免疫抑制的拮抗及其机制
中文摘要:

参照GenBank中安哥斯牛杀菌/通透性增加蛋白(BPI)序列,设计合成1对引物。应用RT-PCR技术,从荷斯坦奶牛嗜中性粒细胞mRNA中扩增出杀菌/通透性增加蛋白N端,将该序列与原核表达载体pGEX-4T-1连接,构建重组质粒pGEX-4T-1-BPI。重组质粒转化大肠杆菌(Escherichia coli)BL21(DE3),用IPTG诱导表达。结果表明,获得了BPIN端长度为714bp的cDNA序列,序列分析证实在第503位出现了点突变。重组质粒诱导表达后,经SDS-PAGE电泳检测重组蛋白约在52kD处出现特异性蛋白条带。

英文摘要:

According to the Bactericidal/permeability-increasing Protein (BPI) sequence of Angus calf in GenBank, a pair of primers were designed. The N-terminal cDNA was amplified by RT-PCR from mRNA which was extracted from the polymorphonuclear neutrophils of Hostein cow. The purified cDNA was connective with expression vector pGEX-4T-1 to construct recombinant plasmid pGEX-4T-1-BPI, which was transformed into Escherichia coli BL21. The results showed that a 714 bp fragment was acquired, and there was a basic mutated group in 503rd compared with the reports. The E.coli with recombinant plasmid was induced with IPTG. The products were analyzed by SDS-PAGE, and a new protein of 52 kD was detected. Its molecular weight was the same as expected, showing the purified recombinant BPI was obtained successfully.

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