牛妊娠相关糖蛋白20真核表达及其结构与功能预测

Eukaryotic expression,structure and function prediction of pregnancy associated glycoprotein 20 in cows

旨在真核表达牛妊娠相关糖蛋白20(bovine pregnancy associated glycoproteins 20,BoPAG20),并对蛋白质结构与功能进行预测。参考GenBank中BoPAG20基因密码子进行优化并合成,构建真核表达载体pcMV3-BoPAG20,转染至293F细胞中进行瞬时表达。SDS-PAGE和Western blot检测表达效果,Ni^(2+)亲和磁珠纯化蛋白,用纯化后的蛋白免疫小鼠验证免疫原性,并对BoPAG20结构与功能进行预测分析。结果:优化后的BoPAG20基因长度为1143 bp,编码380个氨基酸残基,密码子适用指数(CAI)由原来的0.77提高到0.96,GC含量由48.91%提高到55.29%;成功构建了pcMV3-BoPAG20真核表达载体,转染至293F细胞中成功表达,获得BoPAG20蛋白相对分子量约为58 kDa;纯化后BoPAG20蛋白免疫小鼠,34 d后测定血清效价达到1∶10^(5);BoPAG20蛋白无跨膜螺旋区,为分泌性蛋白,信号肽是N端15个氨基酸,存在6个糖基化位点(Ser^(36)、Ser^(56)、Ser^(57)、Ser^(76)、Ser^(80)、Thr^(115))和8个B细胞抗原表位,二级结构由α-螺旋(18.68%)、无规则卷曲(45%)和延伸链(30.79%),无β-转角;与BoPAG20互作蛋白包括β-淀粉样蛋白(APP)和β-淀粉样前体样蛋白2(APLP2)。本研究成功表达并纯化出BoPAG20蛋白,证实其具有良好的免疫原性,并进行了结构和功能预测,为该蛋白的结构和功能研究奠定基础。

In order to expression BoPAG20(bovine pregnancy associated glycoprotein 20)and to predict its structure and function,the codon of the original BoPAG20 gene in GeneBanK was redesigned,optimized and synthesized by bioinformatics techniques.A pcMV3-BoPAG20 expression vector was constructed and was transfected into 293F cells.SDS-PAGE and Western blot were performed to detect the expression of BoPAG20.Then,BoPAG20 was by Ni^(2+)and its immunogenicity was analyzed by immunological test on mice.Finally,the structure and function of the BoPAG20 protein were predicted.The results showed that,after codon optimization,the BoPAG20 gene was 1143 bp long,encoding 380 amino acid residues,that its codon adaptation index(CAI)raised from 0.77 to 0.96 and its G+C content increased from 48.91%to 55.29%.The eukaryotic expression vector pcMV3-BoPAG20 was constructed successfully and expressed in 293F cell,and the protein size was 58 kDa.The purified BoPAG20 protein had good immunogenicity and its antibody titer was as high as 1∶10^(5) in mice for 34 days.BoPAG20 was a secretory protein,with no transmembrane helix.The protein had a signal peptide composed of 15 amino acids,six glycosylation sites(Ser^(36),Ser^(56),Ser^(57),Ser^(76),Ser^(80),and Thr^(115))and eight B cell epitopes.The secondary structure consisted ofα-helix(18.68%),random coil(45%),extended strand(30.79%),respectively,but noβ-turn.The proteins that interacted with BoPAG20 were APP and APLP2.In this study,BoPAG20 protein was successfully expressed and purified,with good immunogenicity,and its structure and function were predicted;which laid a foundation for further research on the structure and function of the protein.