摘要
根据大肠杆菌对精氨酸密码子使用的偏好,设计引物并通过酶促法合成了人β防御素3(hBD-3)全基因序列,克隆进pGEX-4T-2中构建pGEX-4T-2-hBD-3融合表达载体.将表达载体转化E.coli宿主菌DH5α,进行IPTG诱导表达.控制诱导条件,提高可溶性蛋白的表达量.将菌体进行反复冻溶使细胞膜穿孔,释放可溶性蛋白.融合蛋白GST-hBD-3经凝血酶切割得到重组人防御素蛋白.用琼脂孔穴扩散抑菌法检测表明,重组人β防御素3对金黄色葡萄球菌有抑菌活性.
Two oligonucleotide primers were synthesized according to the codon p reference of E.coli to get the whole human beta defensin 3(hBD 3) gene by PCR .The gene was cloned into pGEX 4T 2.Then the recombinant vector pGEX 4T 2 h BD 3 was transformed into E.coli.Clones carrying pGEX 4T 2 hBD 3 was induce d by IPTG.The fusion and soluble induced protein was obtained by repeated cycles of freezing and thawing,then cut by thrombin to get the recombinant hBD 3(rhBD 3).The rhBD 3 showed antibacterial activity to S.aureus by the test of antiba cterial peptide agarose diffusion assay.
出处
《生命科学研究》
CAS
CSCD
2005年第2期129-133,共5页
Life Science Research
基金
广东省自然科学基金资助项目(980174)
关键词
hBD-3
融合表达
反复冻融
hBD-3
fusion expression
repeated freezing and thawing
