牛妊娠相关糖蛋白1(bPAG1)的真核表达及纯化

Eukaryotic Expression and Purification of Recombinant Bovine Pregnancy Associated Glycoprotein-1

[目的]本研究旨在构建PCDNA3.1(-)-bPAG1重组表达载体,并在中国仓鼠卵巢(CHO)细胞细胞中转染表达。[方法]通过基因优化和PCR法扩增得到bPAG1的全基因序列,经T4连接酶将载体PCDNA3.1(-)与酶切bPAG1片段连接,构建PCDNA3.1(-)-bPAG1重组载体后转染到CHO细胞,经SDS-PAGE和Western Blot检测重组牛妊娠相关糖蛋白1(rbPAG1)的表达效果。[结果]PCR扩增得到1218 bp的bPAG1基因片段,构建的重组质粒PCDNA3.1(-)-bPAG1经双酶切获得约5400和1218 bp的2条片段,与预期相符;对重组载体测序,与优化后的基因序列完全一致;SDS-PAGE和Western Blot鉴定显示,获得相对分子质量约为62 kDa的bPAG1重组蛋白,通过Ni柱亲和层析纯化后,rbPAG1纯度可达88%。[结论]本研究为bPAG抗体的制备和奶牛早孕诊断技术的研发奠定了基础。

[Objective]The present paper aimed to construct the eukaryotic expression of PCDNA3.1(-)-bPAGl and express it in Chinese hamster ovary(CHO)cells.[Method]The full-length DNA of bPAGl gene was amplified by PCR and connected with PCDNA3.1(-)vector by T4 DNA Ligase.The PCDNA3.1(-)-bPAGl expression vector was constructed and transfected into Chinese hamster ovary(CHO)cells.SDS-PAGE and western blotting were performed to detect the expression of the recombinant bPAGl(rbPAGl).[Result]The results showed that the optimized sequence of bPAGl gene about 1218 bp was obtained.After amplification and enzymatic digestion,a 1218 bp fragment and a 5400 bp fragment were obtained from PCDNA3.1(-)-bPAGl expression vector.The enzyme digestion and DNA sequencing showed that the DNA sequence of bPAGl gene in PCDNA3.1(-)-bPAGl expression vector was consistent with the nucleotide sequence optimized sequence of bPAGl gene.Western-blotting and SDS-PAGE assays indicated that the rbPAGl with the relative molecular weight of about 62 kDa was successfully expressed in CHO cells.The purity of rbPAGl reached above 88% after purification by Ni^2+ column chromatography.[Conclusion]In this paper,recombinant bPAGl were successfully expressed by eukaryotic expression systems,which laid a foundation for the further study of antibody preparation and dairy cattle early pregnancy diagnosis.