牛妊娠相关糖蛋白9(bPAG9)的真核表达及纯化

【目的】构建proEM-bPAG9重组表达载体,并在HEK293细胞中转染表达。为进一步抗体的制备和奶牛早孕诊断技术的研究奠定基础。【方法】通过基因优化和PCR法扩增得到bPAG9全基因序列,经双酶切法将bPAG9基因插入到表达载体proEM中,构建proEM-bPAG9重组载体后转到DH5α感受态细胞中,重组质粒转染至哺乳动物细胞HEK293中进行瞬时表达,再通过亲和层析纯化bPAG9重组蛋白(rbPAG9),经SDS-PAGE 和 Western Blot 检测rbPAG的表达效果。【结果】通过全基因合成法得到1 182 bp的bPAG9基因片段,构建的重组质粒proEM-bPAG9经双酶切获得约4 369 bp和1 176 bp的两条片段,与预期值相符;对重组载体测序,与优化后的基因序列完全一致,氨基酸未发生突变;SDS-PAGE 和 Western Blot 鉴定显示,获得相对分子质量约为68 kDa 的bPAG9重组蛋白,通过Ni2+亲和层析纯化后,rbPAG9纯度可达90%。【结论】通过基因优化及真核表达获得分子质量为68 kDa 的rbPAG9重组蛋白。

Eukaryotic Expression and Purification of Bovine Pregnancy Associated Glycoprotein-9(bPAG9)

【Objective】 To construct the eukaryotic expression of proEM-bPAG9 and express it in human embryonic kidney (HEK293) cells. 【Method】The full-length DNA of bPAG9 gene was amplified by PCR and connected with proEM vector by T4 DNA Ligase. The proEM-bPAG9 expression vector was constructed and transfected into HEK293 cells. SDS-PAGE and western blotting were performed to detect the expression of the recombinant bPAG9 (rbPAG9). 【Result】The results showed that the optimized sequence of bPAG9 gene about 1,182 bp was obtained. After amplification and enzymatic digestion, a 1,176 bp fragment and a 4,369 bp fragment were obtained from proEM-bPAG9 expression vector. The enzyme digestion and DNA sequencing showed that the DNA sequence of bPAG9 gene in proEM-bPAG9 expression vector was consistent with the nucleotide sequence optimized sequence of bPAG9 gene. Western-blotting and SDS-PAGE assays indicated that the rbPAG9 with the relative molecular weight of about 68 kDa was successfully expressed in HEK293 cells. The purity of rbPAG9 reached above 90% after purification by Ni2+ column chromatography. 【Conclusion】 In this paper, recombinant bPAG9 (68 kDa) were successfully expressed by gene optimization and eukaryotic expression systems, which laid a foundation for the further study of antibody preparation and dairy cattle early pregnancy diagnosis.