非天然氨基酸突变的小鼠RANKL蛋白原核表达及抗血清制备

目的:研究非天然氨基酸突变的小鼠RANKL(m RANKL)蛋白的原核表达,并以表达的蛋白制备抗m RANKL蛋白的抗血清。方法:从小鼠的骨髓中提取总RNA,经PCR扩增m RANKL的胞外段,逆转录合成目的 DNA片段。将目的基因中编码第234位酪氨酸的密码子(TAT)突变成可编码非天然氨基酸p-硝基苯丙氨酸(p NO2Phe)的琥珀密码子(TAG),构建p ET28a-p NO2Phe234m RANKL重组表达载体,与p EVOL质粒共转化至表达菌E.coli BL-21(DE3),诱导表达并纯化。以纯化的蛋白免疫小鼠,制备小鼠抗m RANKL抗血清。采用ELISA测定抗血清效价,用Western Blot测定其特异性。结果:RT-PCR扩增出750bp的RANKL基因,并成功构建了p ET28a-p NO2Phe234m RANKL重组表达载体;p-硝基苯丙氨酸突变的m RANKL蛋白获得成功表达和纯化;以纯化的蛋白免疫小鼠,获得抗m RANKL抗血清,ELISA检测效价为1:6400,Western Blot结果显示该抗血清既可与p-硝基苯丙氨酸突变的m RANKL结合,也可与野生型m RANKL结合。结论:原核表达并纯化了p-硝基苯丙氨酸突变的m RANKL,制备了小鼠抗m RANKL的抗血清,为进一步研究阻断RANKL-RANK通路的新方法奠定了基础。

Prokaryotic Expression of Mouse RANKL with an Unnatural Amino AcidMutant and AntiserumPreparation

Objective:To investigate the prokaryotic expression of mouse RANKL with an unnatural amino acid mutant andpreparation of antiserum.Methods:Total RNA was extracted from mouse bone marrow, and then the extracellular mRANKL gene wasamplified by RT-PCR. PET28a-pNO2Phe234 mRANKL recombinant expression vector was constructed after mutating the 234th genecodon (TAT) encoding tyrosine into the amber codon (TAG) which can encode unnatural amino acid p-nitrophenylalanine (pNO2Phe),and then co-transformed into BL-21 (DE3) with pEVOL plasmid. Protein expression was induced with 0.2 %arabinose and 1 mMIPTG and then purified. Mice were immunized with the purified protein, and the anti-mRANKL antiserum prepared. The antiserum titerwas determined by ELISA, and its specificity was confirmed by Western Blot.Results:mRANKL gene was amplified, and thepET28a-pNO2Phe234 mRANKL recombinant expression vector was successfully constructed; mRANKL protein with ap-nitrophenylalanine mutant was successfully expressed and purified. The antiserum titer was 1: 6400, and Western Blot results showedthat the antiserum could specifically bind not only with p-nitrophenylalanine mutant mRANKL but also with the wild type mRANKL.Conclusion:The protein of p-nitrophenylalanine mutant mRANKL was successfully expressed and purified, and anti-mRANKLantiserum was well prepared, which established the foundation for the further research on new methods of blocking RANKL-RANKpathway.