小鼠载脂蛋白A-Ⅰ的原核表达及抗血清的制备

Prokaryotic Expression of Murine Apolipoprotein A-Ⅰ and Preparation of Its Antiserum

目的原核表达小鼠载脂蛋白A-Ⅰ(ApoA-Ⅰ),并制备抗血清。方法应用RT-PCR方法从小鼠肝脏中扩增ApoA-Ⅰ基因,亚克隆至原核表达载体pET-28a(+),转化大肠杆菌BL21(DE3),IPTG诱导表达。表达的融合蛋白经Ni2+-NTA柱纯化后,免疫家兔,制备抗血清。结果扩增的ApoA-Ⅰ基因经测序,表明与GenBank中报道的序列(NM_009692)完全相同。重组表达质粒经酶切鉴定,证明构建正确。表达的ApoA-Ⅰ融合蛋白相对分子质量约为32000,表达量占菌体总蛋白的17%。纯化的融合蛋白纯度达90.7%,可与抗His·Tag单抗发生特异性反应。制备的抗血清可识别ApoA-Ⅰ融合蛋白,效价可达1∶105。结论已在大肠杆菌中表达了小鼠ApoA-Ⅰ,并制备了较高效价的抗血清。

Objective To express murine apolipoprotein A- I （ApoA- I ） in prokaryotic cells and prepare its antiserum. Methods ApoA- I gene was amplified from the liver tissue of mice by RT-PCR and subcloned into prokaryotic expression vector pET-28a（＋）. The constructed recombinant plasmid pET-28a-ApoA- I was transformed to E. coli BL21 （DE3） for expression under in- duction of IPTG. The expressed fusion protein was purified by Ni2＋-NTA chromatography and used for immunization of rabbits to pre- pare antiserum. Results The sequence of amplified ApoA-I gene was completely identical to that reported in GeuBank （NM_009692）. Restriction analysis proved that recombinant plasmid pET-28a-ApoA- I was constructed correctly. The expressed fusion protein, with a relative molecular mass of about 32 000, contained 17% of total somatic protein, reached a purity of 90. 7% after purification and showed specific reaction with anti-His·Tag MeAb. The prepared antiserurn, at a titer of 1 ： 105, recognized ApoA- I fusion protein specifically. Conclusion Murine ApoA- I was highly expressed in E. coli, and its antiserum at a high titer was prepared.