摘要
目的 构建可生物素化Ｈ－２Ｋ～ｂ－ ＢＳＰ融合基因的表达载体。方法 采用 ＰＣＲ技术，从 ｐＢｌｕｅｓｃｒｉｐｔ（＋） Ｋ～ｂ中扩增出Ｈ－２Ｋ～ｂ基因的胞外区，拼接上依赖 ＢｉｒＡ酶的可生物素化序列后，克隆人ｐＥＴ－４２ｂ（＋）高效表达载体进行诱导表达。并对表达产物进行纯化与复性。结果 成功地构建了融合表达载体ｐＥＴ－Ｈ－２Ｋ～ｂ－ＢＳＰ，对表达产物进行纯化与复性，并应用仅识别完整构象的抗Ｋ～ｂ分子的单克隆抗体对复性后产物进行了检测。证明表达产物已部分恢复了构象。结论 获得Ｈ－２Ｋ～ｂ－ ＢＳＰ融合蛋白的高效表达，为研究在原核系统中表达、纯化与复性，以及进一步利用亲和素在体外构建ＭＨＣ－Ｉ类分子四聚体，探讨免疫识别机制奠定了基础。
Aim To construct expression vector of recombinant H - 2K^b- BSP fusion gene. Methods The extracellular region H - 2K^b gene was amp lified from pBluescript( + )- K^b by PCR, and a 15-a- mino acid substrate peptide for BirA - dependent biotiny lation was added to the COOH- terminus of H- 2K^b heavy chain. Then the fu- sion gene was cloned into pEI- 42b ( +) vector and expressece The expressed fusion protein was pulied and renatured. Results The H- 2K^b fusion protein was successfully expressed and renatured. Con- clusion High - efficient expression of H - 2K^b - BSP fusion protein lays the foundation for further researching expression and purifica- tion in prokaryotic system and constructing tetrameric peptide- MHC complexes to explore the mechanism of immune recognition.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2002年第1期30-32,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助
No.39800132
上海市高校发展基金资助
No.98BJ01.
关键词
H-2K^b
基因表达
纯化
复性
H- 2Kb
gene expression
purification renaburation
