人胱抑素C的原核表达、纯化及多克隆抗体的制备

Prokaryotic expression and purification of human cystatin C and preparation of its polyclonal antibody

目的构建人胱抑素C(Cystatin C,Cys C)基因原核表达质粒,表达并纯化带有硫氧还蛋白(Thioredoxin,Trx)的Trx-Cys C融合蛋白,并制备抗人Trx-Cys C多克隆抗体。方法在不改变氨基酸序列的前提下,对Cys C的全长编码基因进行大肠杆菌同义密码子偏好性优化后,人工合成其序列,插入原核表达载体pET-32a(+)中,构建重组表达质粒pET-32a(+)-Cys C,转化大肠杆菌BL21(DE3),IPTG低温诱导表达。表达的融合蛋白经Ni-Sepharose 6FF纯化后,免疫新西兰大白兔,制备抗人Cys C多克隆抗体,采用间接ELISA及Western blot进行鉴定。结果重组表达质粒经双酶切鉴定构建正确,获得的经同义密码子优化后的序列与预期一致;重组Cys C蛋白获得了可溶性表达,相对分子质量约为35 000,表达量约占菌体总蛋白的50%;纯化的重组蛋白纯度达90%以上,可被Cys C临床检测试剂盒所识别。制备的抗人Cys C多克隆抗体效价达1∶(5.12×106)以上,并能特异性识别市售Cys C蛋白。结论已成功原核表达并纯化了可溶性重组Cys C蛋白,并制备了高效价的抗人Cys C多克隆抗体,为进一步建立Cys C免疫学检测方法奠定了基础。

Objective To construct the prokaryotic expression vector for human cystatin C （Cys C） gene, express and purify thioredoxin （Trx）-Cys C fusion protein, and prepare polyclonal antibody against human Trx-Cys C. Methods A DNA fragment encoding full-length Cys C was designed and synthesized based on optimization of synonymous codon bias of E. coli, without modification of amino acid sequence, and inserted into prokaryotic expression vector pET-32a （＋）. The constructed recombinant plasmid pET-32a（＋）-Cys C was transformed to E. coli BL21 （DE3） and induced with IFI＇G at low temperature. The expressed fusion protein was purified by Ni-Sepharose 6FF chromatography and immunized to New Zealand rabbits, and the prepared polyclonal antibody against human Cys C was identified by indirect ELISA and Western blot. Results Restriction analysis and sequencing proved that recombinant plasmid pET-32a（4-）-Cys C was constructed correctly. The obtained gene sequence with optimized synonymous codon was completely consistent with that expected. Recombinant Cys C protein with a relative molecular mass of about 35 000 was expressed in soluble form, which contained about 50% of total somatic protein. The purified recombinant protein reached a purity of more than 90%, and was recognized by clinical detection kit for Cys C. The prepared polyclonal antibody reached a titer of more than I ： （5. 12 × 10^6）, and recognized commercial Cys C protein specifically. Conclusion Soluble recombinant Cys C protein was successfully expressed in prokaryotic cells, and its polyclonal antibody with high titer was prepared, which laid a foundation of development of immunological method for detection of Cys C.