G17-白喉毒素嵌合蛋白的表达、纯化和免疫原性初步研究

目的  构建胃泌素多肽G17-白喉毒素A亚单位（diphtheria toxin subunit A，DTA）嵌合蛋白，并初步研究其免疫原性。方法  用酶切法将编码G17的基因与DTA基因连接后，插入载体pET11C，并在大肠埃希菌BL21中表达。先后用阴离子交换色谱法和分子排阻色谱法对表达的目的蛋白进行纯化。用纯化的G17-DTA免疫NIH小鼠，并用ELISA检测小鼠抗G17抗体。结果  构建的G17-DTA嵌合蛋白可在大肠埃希菌中高效表达，表达量达0.5~0.6 mg/ml菌液。经2次纯化后，G17-DTA的纯度可达95%。纯化的G17-DTA在小鼠中诱生了抗G17抗体。结论  构建的G17-DTA嵌合蛋白能在大肠埃希菌中表达，而且在小鼠中具有免疫原性。

Expression,purification and preliminary immunogenicity study of chimeric protein G17-diphtheria toxin subunit A

Objective  To construct chimeric protein gastrin peptide G17-diphtheria toxin subunit A (DTA), and preliminarily study its immunogenicity. Methods  After G17-coding gene was linked with DTA gene using enzyme digestion, G17-DTA fragment was inserted into vector pET11C and expressed in E.coli BL21. The expressed protein was purified using anion exchange chromatography followed by molecular exclusion chromatography. NIH mice were immunized with the purified G17-DTA, and anti-G17 antibody was detected by ELISA. Results  The constructed chimeric protein G17-DTA was highly expressed in E.coli. The amounts of protein were 0.5-0.6 mg/ml bacterial solution. The purity of G17-DTA achieved 95% after 2 purifications. NIH mice immunized with G17-DTA produced anti-G17 antibodies. Conclusion  The constructed chimeric protein G17-DTA is successfully expressed in E.coli, and has immunogenicity in mice.