兔抗人ERA抗血清的制备

Preparation of rabbit- anti human ERA serum

目的在大肠杆菌中表达人ＥＲＡ蛋白（ｈ－ＥＲＡ）和ｈ－ＥＲＡ Ｃ端结构域蛋白，并制备兔抗ｈ－ＥＲＡ抗血清。方法以 ＰＣＲ扩增人ｅｒａ基因（ｈ－ｅｒａ）全长ｃＤＮＡ和 ｈ－ＥＲＡ Ｃ端的结构域基因，并分别克隆到（Ｈｉｓ）６融合表达载体ＰＲＳＥＴ－Ｃ和非融合表达载体ｐＤＨ中，诱导表达（Ｈｉｓ）６－ｈ－ＥＲＡ融合蛋白和ｈ－ＥＲＡ Ｃ端结构域蛋白。用 ｈ－ＥＲＡ Ｃ端结构域蛋白免疫兔，制备兔抗ｈ－ＥＲＡ抗血清，并以Ｗｅｓｔｅｒｎ－ｂｌｏｔ对免抗ｈ－ＥＲＡ抗血清进行鉴定。结果大肠杆菌中表达的ｈ－ＥＲＡ Ｃ端结构域蛋白和（Ｈｉｓ）６－ｈ－ＥＲＡ融合蛋白，分别占菌体总蛋白的４０％和８０％。用兔抗血清可检出大肠杆茵中表达的（Ｈｉｓ）６－ｈ－ＥＲＡ融合蛋白。结论 ｈ－ＥＲＡ和 ｈ－ＥＲＡ Ｃ端结构域蛋白在大肠杆菌中获得高效表达，并成功地制备了兔抗ｈ－ＥＲＡ抗血清。

Aim To express h-ERA and h-ERA C-terminal domain in E. coli and prepare antiserum against human ERA. Methods Human era(h-era) cDNA and h-ERA C-terminal domain gene were amplified by PCR and ligated with prokaryotic expression vector pRSET-C and pDH respectively. E. coli TAP106 transformed with the recombinant plasmid pDH-h-era-C was induced at 42℃ to express h-ERA C-terminal domain protein. Antiserum against h-ERA was prepared by immunizing rabbit with h-ERA C-terminal domain protein purified by SDS-PAGE. E. coli BL21 (DE3) transformed with the recombinant plasmid pRSET-C-h-era was induced with IPTG to express (His)6-h-ERA fusion protein for Western-blot analysis. Results The expressed h-ERA C-terminal domain and (His)6-h-ERA fusion protein occupied about 40% and 80% of total bacterial protein respectively. The(His) 6-h-ERA fusion protein expressed in E. coli can be detected with the rabbit antiserum. Conclusion The h-ERA C-terminal domain protein and (His)6-h-ERA fusion protein were expressed with high efficiency in E. coli, and the rabbit antiserum against h-ERA was prepared successfully.