摘要
目的克隆并表达牛乳铁蛋白十肽及其突变体M1和M2基因。方法参照大肠杆菌偏爱密码子,分别针对牛乳铁蛋白十肽及其突变体M1和M2基因,设计并合成两个具有相同黏性末端的DNA片段,同时在其基因前后分别加上天冬酰胺和甘氨酸的密码子,构成羟胺裂解位点,通过连接获得其二拷贝基因同向串联体。分别将该串联体克隆至载体pUC18上,经双酶切、PCR及测序鉴定后,构建重组表达质粒,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物纯化后,用凝血酶切割及羟胺裂解。结果获得了牛乳铁蛋白十肽及其突变体M1和M2二拷贝基因,并在大肠杆菌BL21(DE3)中表达出相对分子质量约29000的目的蛋白条带,纯化后的蛋白经凝血酶切割后,相对分子质量约29000的目的蛋白条带消失。结论已成功克隆并表达了牛乳铁蛋白十肽及其突变体M1和M2基因,为基因工程抗真菌肽的制备奠定了基础。
Objective To clone and express the genes encoding bovine lactoferrin decapeptide and its mutants （MI and M2）. Methods Two DNA fragments with the same sticky ends, containing E.coli-preferred asparamide and glycine codons as well as hydroxylamine cleavage site, were designed according the amino acid sequences of bovine lactoferrin decapeptide and its mutants M1 and M2 respectively, and synthesized chemically, then linked to obtain two-copy gene tandem repeats. Clone the tandem repeats into vector pUC18 respectively and transform the constructed recombinant plasmid, after identification by restriction analysis, PCR and sequencing, to E. coli BL21 （DE3） for expression under induction of IPTG. The expressed product was purified with B-PER GST SpinPurification kit and cleaved with hydroxylamine. Results The two-copy gene tandem repeats of bovine lactoferrin decapeptide and its mutants were obtained, and the target protein with a relative molecular mass of about 29 000 was expressed in E. coli BL21 （DE3）. After the purified expressed product was digested with thrombase, the protein band with a relative molecular mass of about 29 000 disappeared on SDS-PAGE profile. Conclusion The gene encoding bovine lactoferrin decapeptide and its mutants were successfully cloned and expressed, which laid a foundation of preparation of recombinant antifungal peptide.
出处
《中国生物制品学杂志》
CAS
CSCD
2008年第7期568-571,共4页
Chinese Journal of Biologicals
关键词
牛乳铁蛋白十肽
抗真菌肽
基因改造
基因克隆
表达
Bovine lactoferrin decapeptide
Antifungal peptide
Genetic modification
Gene cloning
Expression
