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目的表达和纯化带多聚组氨酸(6×His)标签的人Tenascin-R EGFL结构域(Tenascin-Raa454-1069)的融合蛋白,并制备相应的兔源性多克隆抗体(pAb)。方法利用PCR从PMD18-T-TNR获得Tena-scin-Raa454-1069编码序列,将其亚克隆至原核表达载体pET30a(+),构建重组表达质粒pET30a(+)-Tenascin-Raa454-1069;将阳性重组质粒转化大肠杆菌,IPTG诱导表达6×His-Tenascin-Raa454-1069融合蛋白,经Ni-NTA螯合树脂纯化,纯化蛋白免疫新西兰大白兔制备多克隆抗血清,Protein A Sepharose柱纯化获得多抗,ELISA法检测抗体效价,Western blot法检测抗体特异性。结果成功构建了pET30a(+)-Tenascin-Raa454-1069原核表达载体,原核蛋白Tenascin-Raa454-1069以包涵体形式在大肠杆菌高水平表达,通过复性与亲和层析获得纯度在90%以上的融合蛋白,蛋白浓度为1 600 mg/L,制备的抗Tenascin-Raa454-1069多抗效价高达1∶1.6×106,Western blot鉴定其具有良好的特异性。结论获得高纯度Tenascin-Raa454-1069蛋白并成功制备特异性pAb,为进一步研究Tenascin-R的生物学功能提供实验基础。
Objective To express and purify the fusion protein of human Tenascin - R EGFL domain (Tenascin - Raa454-1069) with polyhistidine (6 × His) tag and prepare the corresponding rabbit polyclonal antibody (pAb). Methods Tena-scin-Raa454-1069 coding sequence was obtained from PMD18-T-TNR by PCR and subcloned into prokaryotic expression vector pET30a (+) to construct recombinant expression plasmid pET30a (+) - Tenascin-Raa454-1069. Recombinant plasmids were transformed into E.coli. IPTG was used to induce the expression of 6 × His-Tenascin-Raa454-1069 fusion protein. The purified protein was purified by Ni-NTA chelating resin and immunized New Zealand white rabbits to prepare polyclonal antiserum. Protein A Sepharose column was used to obtain polyclonal antibody The antibody titer was detected by ELISA and the antibody specificity by Western blot. Results The prokaryotic expression vector pET30a (+) - Tenascin-Raa454-1069 was constructed successfully. The prokaryotic expression protein Tenascin-Raa454-1069 was expressed in high level in the form of inclusion bodies in Escherichia coli. The purity was over 90% by renaturation and affinity chromatography Of the fusion protein at a protein concentration of 1 600 mg / L. The titer of the prepared anti-Tenascin-Raa454-1069 polyclonal antibody was as high as 1: 1.6 × 10 6, and its specificity was confirmed by Western blot. Conclusion The high-purity Tenascin-Raa454-1069 protein was obtained and the specific pAb was successfully prepared, which provided the experimental basis for further study on the biological function of Tenascin-R.