牛乳素Lfcin B基因的合成及其在酵母菌中的表达研究

目的]为临床研究和治疗某些病原微生物引起的感染提供依据。方法]参照LfcinB的氨基酸序列,利用化学合成法合成LfcinB基因并克隆到pPIC9K载体中,转化到酵母菌GS115感受态细胞中,用抗性选择标记G418筛选出高拷贝酵母菌转化子。利用甲醇诱导表达重组LfcinB,经Tricine-SDS-PAGE电泳检测目的蛋白牛乳铁蛋白素的表达情况,同时通过体外抑菌试验检测表达产物的抑菌活性。结果]提取的酵母菌DNA扩增出1条预期的560bp左右的特异性条带。目的基因已经成功克隆到pPIC9K载体中,并转到酵母菌GS115中而且获得了表达。重组酵母表达上清中牛乳铁蛋白素抗菌肽对沙门氏肠炎杆菌的抗菌活性为42.857IU/ml,表明LfcinB基因的酵母表达载体构建成功。结论]该研究证明利用化学合成法直接合成编码LfcinB的DNA构建真核表达载体、通过生物发酵工程来制备LfcinB是可行的。

Study on the Synthesis of Lactoferricin Bovine(Lfcin B)Gene and Its Expression in Microzyme

Objective] The research aimed to provide basis for clinical research and cure the infection caused by some pathogenic microorganisms.Method] According to the amino acid sequence of Lfcin B, the gene was synthesized by using chemical synthesis method and cloned into pPIC9K vector. Then it was transformed into competent cells of microzyme GS115 and high-copy microzyme transformants were screened out by using resistant selective marker G418. The expression of the recombinant Lfcin B gene was induced by methanol and the expression conditions of target protein Lfcin B were detected by Tricine- SDS- PAGE and at the same time the antibacterial activity of expression product was detected by bacteriostatic test in vitro. Result] An expected specific band with the length of about 560 bp was amplified by the extracted microzyme DNA. The target gene was successfully cloned into pPIC9K vector and transformed into microzyme GS115 for obtaining expression. The antibacterial activity of antibacterial peptide of Lfcin B in expression supernatant of the recombinant yeast to Salmonella enteritidis was 42.857 IU/ml, which indicated that yeast expression vector of Lfcin B gene was constructed successfully. Conclusion] This research proved that it was feasible to construct eukaryotic expression vector by directly synthesizing DNA coded by Lfcin B by using chemical synthesis method and preparing Lfcin B by biological fermentation engineering.