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

Cloning and Expression of Fused Fc-binding Protein(SPA-SPG) and Its Application in Purification of IgG

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T4DNA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 1h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.