中文摘要：

目的表达和纯化带多聚组氨酸（6×His）标签的人Tenascin—REGFL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Tenascin-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1600mg／L，制备的抗Tenascin-Raa454-1069多抗效价高达1：1．6×10^6，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。

英文摘要：

Objective To express and purify the fusion protein of EGFL region of human Tenascin - R （hTenascin-Raa454-1069） in prokaryocytes and to prepare rabbit anti - Tenascin-Raa454-1069 polyclonal antibody （pAb）. Methods The 616 bp DNA sequence of hTenascin-Raa454-1069 was obtained from PMD18 -T -TNR by PCR and subsequently inserted into pET30a（ ＋ ） plasmid to construct the prokaryotic expression plasmid pET30a（ ＋ ） -hTenascin-Raa454-1069. pET30a（ ＋ ） - hTenascin-Raa454-1069 was transformed into E. coli. The target fusion protein was expressed with the induction of Isopropyl 13 -D- 1 -thiogalactopyranoside （IPTG） and was purified by Ni^2＋ -NTA affinity chromatography col- umn. The antiserum against hLINGO- 1aa76-319 was obtained from the rabbits immunized with hTenascin-Raa454-1069. The titer of pAb was determined by ELISA and the specificity of pAb was confirmed by Western blot. Results The prokaryotic expression plasmid pET30a（ ＋ ） -hTenascin-Raa454-1069was successfully constructed. The culture condition for high expression of target fusion protein was as followings： The optimal concentration of IPTG was 0.4mmol/L, the culture temperature was 37~C and the culture time was 2.5 h. The hTenascin-Raa454-1069 fusion protein was effectively expressed in E. coli as inclusion body. Soluble protein was obtained through denaturation and refolding procedure. The fusion protein was purified by Ni - NTA Resin affinity column was more than 90% purity. The anti - hLINGO - 1aa76-319 pAb was prepared by immunizing the rabbits with the purified target fusion protein. ELISA showed the titer of pAb was 1 ： 1.6 × 10^6. Westeru blot analysis confirmed anti - hLINGO - 1aa76-319 pAb was of good specificity. Conclusion The fusion protein hTenascin - Raa454-1069 with high purity has been obtained and the anti - hTenascin-Raa454-1069 pAb with high titer and good specificity has been prepared. Our study facilitates further research into the molecular function of Tenascin - R.