CCL3L1融合蛋白的表达纯化及其多克隆抗体的制备

Expression and purification and activity assay of CCL3L1 fusion protein

目的表达和纯化CCL3L1融合蛋白,并对其免疫原性进行分析。方法应用分子生物学技术将pGEX-4T-1-CCL3L1质粒进行酶切,收集CCL3L1片段与pET-32a(+)表达载体连接,构建pET-32a(+)-CCL3L1重组质粒,将其转化BL-21大肠埃希菌进行蛋白表达并纯化。应用酶切鉴定、SDS-PAGE及Western blot等方法确保基因片段的正确性及表达蛋白的特异性。以间接ELISA法测定BABL/c小鼠多克隆抗体滴度。结果成功获得了高纯度的CCL3L1融合蛋白,且该蛋白为可溶性表达,以其制备的多克隆抗体滴度最高可达1∶51200。结论获得高纯度可溶性表达的CCL3L1融合蛋白及其高效价的多克隆抗体。

Objective To express and purify CCL3L fusion protein and test its immunoactivity. Methods Recombinant plasmid pET-32a（ ＋ ）-CCL3L1 was constructed by inserting CCL3L1 gene fragment which was digested with BamH I and Xho I from pGEX-4T-1-CCL3L1 plasmid, into the expressive vector pET-32a（ ＋ ）. The recombinant prokaryotic expression plasmid, namely pET-32a（ ＋ ） -CCL3L1, was transformed into Escherichia coli strain BI2.1 （DE3）. Subsequently, fusion protein was expressed efficiently in prokaryotic system through IPTG induction and purified by Ni2＋ -NAT. The pET-32a（ ＋ ）-CCL3L1 fusion protein could specifically react with His antibody and anti-CCL3L1 polyclonal antibody by Western blot technique. The product was also analyzed by SDS-polyacrylamide gel electrophoresis and a protein about 27 kD was observed. Using the purified fusion protein, we immunized female BALB/c mice by subcutaneous injection three times. Serum samples were tested by an enzyme-linked immunosorbent assays （ELISAs） to determine the level of antibodies. The result showed that fusion protein could stimulate animals to produce special and sensitive antibody. Results pET-32a（ ＋ ）-CCL3L1 fusion protein mainly exist solubly and its purity was above 80%. The titer of the antibodies was 1 ： 51 200. Conclusions pET-32a（ ＋ ） -CCL3L1 fusion protein with high purity was obtained and its corresponding polyclonal antibodies with high titer were produced.