摘要
目的为提高杀菌/渗透性增强蛋白N端功能片段BPI_(23)对内毒素(lipopolysaccharides,LPS)的亲和性,通过易错聚合酶链式反应(polymerase chain reaction,PCR)和DNA改组技术,对其编码序列BPI_(600)进行突变,在大肠杆菌XL10-Gold中构建BPI_(600)基因突变体库。方法通过控制Mg2+和Mn2+的浓度进行易错PCR,获得BPI_(600)随机突变基因片段,经测序和基因比对,确定BPI_(600)的随机突变率。再对易错PCR产物进行DNA改组,将改组产物克隆至pYD1载体中,转化大肠杆菌XL10-Gold,从Amp抗性(LuriaBertani,LB)固体培养平板上任选10个单菌落,增菌后提取质粒,得到pYD1-shuffled BPI_(600)重组质粒,将质粒进行HindⅢ/XhoⅠ酶切鉴定,取5个阳性质粒进行DNA测序,通过基因和氨基酸比对鉴定突变情况。结果测序和基因比对显示,BPI_(600)经易错PCR所获突变文库的随机突变率达2.3%;改组后重组质粒的酶切结果表明,在随机选择的10个菌落所提质粒中,有6个含BPI_(600),表明构建了重组质粒pYD1-shuffled BPI_(600);在5个阳性质粒中,有4个重组质粒的BPI_(23)编码序列分别发生了6、9、11和14个碱基突变;1个不仅有10个碱基突变,还存在1个碱基的缺失。氨基酸比对表明,有2个质粒(分别有6和14个碱基突变)具有正确的读码框,能够翻译完整的蛋白质,各有4或14个氨基酸突变。3个质粒因含终止密码子而不能表达完整目的蛋白。结论成功构建pYD1-shuffled BPI_(600)重组质粒,获得库容量为2×105的突变文库,为高内毒素亲和力突变体的筛选奠定了基础。
Objective To construct and identify a gene mutation library of the N-terminal fragments of human bactericidal/permeability increasing proteins（BPI_（23）） for enhancing its activity binding lipopolysaccharides（LPS） by error-prone polymerase chain reaction（PCR） and DNA shuffling method.Methods Error-prone PCR was used to obtain random mutant fragments of BPI_（600） by adding different concentrations of Mg^（2＋） and Mn^（2＋）.Random mutation rate was calculated by DNA sequencing and genetic comparison with wildtype BPI_（600）.The product of error-prone PCR was further mutated via DNA shuffling.The reassembled product was cloned into pYD1 vector and transformed into E.coli XL10-Gold.Ten clones were randomly picked out,digested and identified by restriction enzyme HindⅢ and XhoⅠ.The positive clones were identified by DNA sequencing.Results DNA sequencing result showed that the random mutation rate was 2.3% by error-prone PCR.After DNA shuffling,6 positive clones including BPI_（600） were identified in the 10 random clones selected fromLB culture medium with ampicillin.Among them,4 had 6,9,11 and 14 mutations,respectively;1 had 10 mutations and 1 deletion determined by DNA sequencing.By amino acid comparison,only 2 DNA mutants were able to encode a full-length mutant BPI_（23） protein,with 4 and 14 amino acid mutations respectively.Conclusion A library of BPI_（23） mutants was constructed with a size of 2×10~5.The results laid the foundation for the study of its activity binding LPS.
出处
《首都医科大学学报》
CAS
北大核心
2017年第6期903-910,共8页
Journal of Capital Medical University
关键词
人杀菌
渗透增强蛋白
定向进化
易错PCR
DNA改组
human bactericidaL/penneability increasing protein
directed evolution
error-prone polymerase chain reaction（P C R ）
DNA shuffling
