云南旺源生物科技有限公司资助项目.
This work was supported by Yunnan Wangyuan Biotech CO., LTD.
利用生物信息学,遴选编码SPA及SPG蛋白的基因,进行密码子优化,将目的基因分割成互为重叠的小片段寡聚核苷酸链,采用一步升温后T4 DNA连接酶连接的方法,合成了编码SPA及SPG蛋白的融合基因.将其克隆到pSK质粒进行扩增, 经测序、修正后再克隆到表达载体,高效表达了带His6的融合蛋白——蛋白AG.将蛋白AG共价结合到表面带羧基的磁粒上,形成蛋白AG磁粒复合物,用此复合物可在1 h内从大鼠、小鼠、人、猕猴、马、羊及猪等常用实验动物血清中纯化IgG.
Using a bioinformatical approach, a hybrid gene encoding the Fc-binding domains of both SPA and SPG (1 269 bp) was designed and synthesized. The sequence was designed by substitution of high-usage codons for low-usage ones of Escherichia coli. A total of 64 oligonucleotides were assembled in a single-step, in which T4 DNA ligase ligated DNA fragments from a pool of overlapping oligonucleotides. The synthetic gene was cloned to pSK vector. After sequencing and remending, the gene was expressed using the expression vector pET-28a-c(+). A band of 47 ku was detected with Western blot analysis. The protein so called MproteinAG has been covalently conjugated to the carboxyl-terminated magnetic particles. With the MproteinAG magnetic particles,IgG from blood serium of rabbit, pig,dog,mouse, human, macaque, rat, cat, sheep, cattle, horse can be purificated in one hour.
孟照辉,林兵,谢月辉,张克勤. Fc段受体结合蛋白(SPA-SPG)基因克隆表达及其在IgG纯化中的应用[J].生物化学与生物物理进展,2004,31(2):146-149
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