人体β-防御素-3突变基因构建及在大肠埃希菌中融合表达.docVIP

人体β?防御素?3突变基因构建及在大肠埃希菌中融合表达 　 【摘要】; 目的 从人正常皮肤组织中提取β?防御素?3基因进行定点突变后在E.coli中融合表达。方法 从人正常皮肤组织中提取总RNA，经RT?PCR扩增得到编码β?防御素?3成熟肽的cDNA序列，测序正确后，寻找合适的突变位点，设计含突变位点的引物，用PCR重叠延伸法对β?防御素?3成熟肽cDNA序列进行定点突变，将突变产物克隆至pUC18进行序列测定及在E.coli中融合表达。结果 测序结果表明，经RT?PCR所得的β?防御素?3成熟肽序列与GeneBank中报道的编码人β?防御素?3成熟肽的cDNA序列完全一致。经过突变β?防御素?3成熟肽第29位谷氨酰胺密码子由CAG突变为精氨酸密码子CGA，其余核苷酸序列均未发生变化。将突变β?防御素?3基因在E.coli中诱导表达3～5h后可见突变β?防御素?3蛋白表达。结论 成功构建并表达了β?防御素?3突变体基因，为进一步对突变体进行真核表达、生物活性研究奠定了基础。 毕业论文 【关键词】; 人体β?防御素?3； 定点突变； 基因克隆； 测序； 基因表达 毕业论文 　　Construction and expression of a site?directed mutant gene　of human beta?defensin?3 毕业论文 　　ABSTRACT; Objective; To extract the gene encoding human beta?defensin?3 (hBD?3) from human foreskin tissue, make a site?directed mutagensis and expression with the mutant gene in E.coli.; Methods; The total RNA extracted from human foreskin tissue and the sequence of cDNA encoding hBD?3 was amplified by RT?PCR. After sequencing, a pair of mutant primers was designed and site?directed mutation was made by over?lap extension PCR and then the mutant gene was expressed in E.coli.; Results; Sequencing analysis indicated that hBD?3 DNA obtained by RT?PCR was identical to the sequence reported in GenBank. After mutation, the code CAG for glutamine was changed into the code CGA for arginine at position 29. After induction for 3～5 hours, a new protein band appeared on SDS?PAGE gel.; Conclusion; Mutant hBD?3 (mhBD?3) gene is constructed and expressed in E.coli BL21 successfully. It might provide a foundation for construction of eukaryotic expression vector and research of its biological activity. 毕业论文 ; 　　KEY WORDS; Human beta?defensin?3 Site?directed mutagensis Gene cloning Sequencing Gene expression 毕业论文 　　抗菌肽是生物体产生的一类抗微生物的阳离子小分子多肽，是生物天然免疫的重要组成部分［1］。由于这些物质对许多致病菌都有抗菌作用，而且无致畸作用，无蓄积毒性，最为重要的是在机体内不易产生耐药性，因此有望开发为新型的抗生素，以解决日益严重的传统抗生素耐药性问题。 ; 　　 　　防御素(defensin)是近年来发现的一组富含半胱氨酸残基的抗菌肽，主要存在于上皮组织中，构成机体抵御病原微生物侵袭的第一道化学屏障［2］。人体β?防御素?3(human beta?defensin?3，hBD?3)是新近被克隆出来的第三种人源性β?防御素。它主要对革兰阳性菌