| 李锋 李寰 沙鑫 杨卫周 马文瑞 陶惠人.非天然氨基酸突变的小鼠RANKL蛋白原核表达及抗血清制备[J].现代生物医学进展英文版,2015,15(3):437-440. |
| 非天然氨基酸突变的小鼠RANKL蛋白原核表达及抗血清制备 |
| Prokaryotic Expression of Mouse RANKL with an Unnatural Amino AcidMutant and AntiserumPreparation |
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| DOI： |
| 中文关键词: 非天然氨基酸 RANKL 原核表达 抗血清 |
| 英文关键词: Unnatural amino acid RANKL Prokaryotic expression Antiserum |
| 基金项目:国家自然科学基金项目（81070698；81170256）；陕西省科学技术研究发展计划项目（2009K13-01） |
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| 中文摘要: |
| 目的：研究非天然氨基酸突变的小鼠RANKL（mRANKL）蛋白的原核表达，并以表达的蛋白制备抗mRANKL蛋白的抗血
清。方法：从小鼠的骨髓中提取总RNA，经PCR 扩增mRANKL 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（pNO2Phe）的琥珀密码子（TAG），构建pET28a-pNO2Phe234
mRANKL 重组表达载体，与pEVOL质粒共转化至表达菌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mRANKL 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pET28a-pNO2Phe234mRANKL重组表达载体；p- 硝基苯丙氨酸突变的mRANKL 蛋白获得成功表达和纯化；以纯化
的蛋白免疫小鼠，获得抗mRANKL 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mRANKL结合，也可与野生型mRANKL 结合。结论：原核表达并纯化了p- 硝基苯丙氨酸突变的mRANKL，制备了小鼠
抗mRANKL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。 |
| 英文摘要: |
| Objective:To investigate the prokaryotic expression of mouse RANKL with an unnatural amino acid mutant and
preparation of antiserum.Methods:Total RNA was extracted from mouse bone marrow, and then the extracellular mRANKL gene was
amplified by RT-PCR. PET28a-pNO2Phe234 mRANKL recombinant expression vector was constructed after mutating the 234th gene
codon (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 mM
IPTG and then purified. Mice were immunized with the purified protein, and the anti-mRANKL antiserum prepared. The antiserum titer
was determined by ELISA, and its specificity was confirmed by Western Blot.Results:mRANKL gene was amplified, and the
pET28a-pNO2Phe234 mRANKL recombinant expression vector was successfully constructed; mRANKL protein with a
p-nitrophenylalanine mutant was successfully expressed and purified. The antiserum titer was 1: 6400, and Western Blot results showed
that 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-mRANKL
antiserum was well prepared, which established the foundation for the further research on new methods of blocking RANKL-RANK
pathway. |
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