基于密码子优化策略的牛妊娠相关糖蛋白9(bPAG9)的原核表达及纯化

Prokaryotic expression and purification of recombinant bovine pregnancy associated glycoprotein-9 (bPAG9) based on codon optimization

为了构建pET30a-bPAG9重组表达载体,并在BL21(DE3)感受态细胞中转染高效表达牛妊娠相关糖蛋白9(bPAG9),通过生物信息学技术对bPAG9基因进行密码子优化,人工合成bPAG9全基因序列,经双酶切法将bPAG9基因插入到表达载体pET30a中。将构建的重组质粒pET30a-bPAG9转染至BL21(DE3)感受态细胞中进行原核表达,经过超声波裂解和亲和层析方法获得重组蛋白bPAG9。用SDS-PAGE和Western Blot方法检测重组蛋白bPAG9的表达效果。结果显示,PCR扩增得到1128 bp的优化bPAG9基因片段,构建的重组质粒pET30a-bPAG9经双酶切获得约5244 bp和1125 bp的2条片段,与预期值相符;对重组载体测序,测序结果与优化后的基因碱基序列完全一致,编码的氨基酸序列未发生突变;SDS-PAGE和Western Blot鉴定结果显示,获得相对分子质量约为4.0×10^4的bPAG9重组蛋白,通过亲和层析纯化后,重组蛋白bPAG9纯度到达90%以上。

To construct the recombinant expression vector pET30a-bPAG9 and express bovine pregnancy associated glycorprotein-9(bPAG9)in BL21(DE3)cells,the codons of original bPAG9 gene were redesigned and optimized by bioinformatics techniques.Furthermore,the codon-optimized gene bPAG9 was synthesized by PCR and connected with pET30a vector by double digestion.The pET30a-bPAG9 expression vector was constructed and transfected into BL21(DE3)cells.The recombinant protein bPAG9 was obtained by ultrasonic lysis and affinity chromatography.SDS-PAGE and Western blotting were performed to detect the expression of the recombinant protein bPAG9.The results showed that the optimized bPAG9 gene fragment of 1128 bp was obtained by PCR.After amplification and enzymatic digestion,two fragments of 5244 bp and 1125 bp were obtained from pET30a-bPAG9 expression vector.These results were consistent with expectations.The sequencing results of recombinant vector showed that the base sequence of bPAG9 gene in expression vector was consistent with the optimized gene,and the amino acid was not mutated.The results Westernblotting and SDS-PAGE indicated that the recombinant protein bPAG9 with the relative molecular weight of about 4.0×10^4 was successfully expressed in BL21(DE3)cells.The purity of recombinant protein bPAG9 was over 90%after purification by affinity chromatography.