兔抗多疣壁虎Hoxc10抗血清的制备及鉴定  

The preparation of rabbit antiserum against gekko japonicus Hoxc10 and it’s property identification

作　　者：周晓芳[1] 陆仁飞[2] 陈倩[1] 储钰丹[1] 王学谦[1] 钱天梅[1] 顾星星[1] 

机构地区：[1]南通大学神经再生重点实验室,江苏南通226001 [2]南通市第三人民医院检验科,江苏南通226001

出　　处：《细胞与分子免疫学杂志》2011年第10期1110-1113,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30671046;31071288);南通大学博士基金项目(07B21)

摘　　要：目的:制备兔抗多疣壁虎Hoxc10抗血清并进行特性鉴定。方法:构建pGEX-4T1-g-Hoxc10质粒,转化大肠杆菌BL21后,在异巯基-1-硫代-β呋喃半乳糖(IPTG)诱导下,表达谷胱甘肽S-转移酶(GST)-g-Hoxc10蛋白。用亲和层析纯化的融合蛋白免疫大白兔,制备Hoxc10抗血清。用Western blot和免疫组化染色法检测Hoxc10抗血清的特性。结果:构建的多疣壁虎Hoxc10基因的原核表达质粒pGEX-4T1-g-Hoxc10,在大肠杆菌中获得高效表达。表达产物经亲和层析纯化后得到高纯度的Hoxc10融合蛋白,以其免疫大白兔获得较高效价的Hoxc10抗血清。Western blot及免疫组化染色法检测显示,所得抗血清具有较高的效价及特异性。结论:利用原核表达的多疣壁虎GST-g-Hoxc10融合蛋白制备的Hoxc10抗血清效价高,特异性良好,为进一步深入研究Hoxc10的功能提供了重要的制剂。AIM： Prepare the rabbit antiserum against gecko japonicus Hoxc10 and to identify its properties. METHODS： Prokaryotic expression vector of g-Hoxc10 were constructed and then transform into E.coli （BL21）. To make GST-g-Hoxc10 fusion protein in E.coli （BL21） under the optimized induction of Isopropyl β-D-1-thiogalactopyranoside（IPTG）. The recombination proteins were purified using affinity chromatography. The purified fusion protein was inoculated into adult rabbits to develop antiserum. Western blot and immunohistochemistry staining were then performed to evaluate the feature of the prepared antiserum. RESULTS： Prokaryotic expression vectors of g-Hoxc10 were successfully constructed. The soluble recombinant protein was highly expressed in E.coli BL21 and inoculated into adult rabbits to obtain high titer antiserum. Western blot and immunohistochemistry staining were then performed to evaluate the specificity of the prepared antiserum. CONCLUSION： We successfully amplified and expressed the g-Hoxc10 in E.coli BL21. The purified fusion protein was inoculated into adult rabbits to develop antiserum. The obtained antiserum of g-Hoxc10 showed a high titer against Hoxc10 proteins. The protein and antiserum prepared in this study can be used for further research of the function investigation of Hoxc10.

关 键 词：多疣壁虎 Hoxc10 融合蛋白 抗血清 兔 

分 类 号：R392.11[医药卫生—免疫学]