摘要
目的原核表达小鼠脂多糖应答蛋白并制备兔抗mLRP抗血清。方法参考GenBank中已登录的人lrp-cDNA序列,对小鼠的同源表达序列标签(EST)序列进行拼接,预测mlrpcDNA序列。用设计的引物,进行RT-PCR,从脂多糖刺激的NIH3T3细胞中扩增mlrp的cDNA序列,克隆入pTAT载体中,构建mlrp的原核表达载体。以其转化在大肠杆菌中诱导表达后,将获得的His-TAT-mLRP融合蛋白免疫家兔,用丙酮沉淀全菌蛋白吸附法制备纯化的抗mLRP血清,用West-ernblot鉴定抗体的特异性。结果预测的mlrpcDNA的长度为1905bp。将克隆获得的1554bp大小的mlrp-cDNA编码区序列插入pTAT质粒中,在大肠杆菌中表达出His-TAT-mL-RP融合蛋白。以其免疫家兔,制备了特异性兔抗mLRP的血清。结论mlrp的原核表达及特异性兔抗mLRP血清的制备,为进一步研究mLRP的功能奠定了基础。
AIM： To express mouse lipopolysaccharide response protein（mLRP） and prepare rabbit anti-mLRP serum. METHODS： The predicted mouse lrp cDNA sequence was obtained by splicing homologous ESTs by comparing human lrp cDNA with mouse ESTs, Then the primers were designed, mlrp cDNA from NIH3T3 cells stimulated with lipopolysaccharide （LPS） was amplified by RT-PCR and was cloned into prokaryotic expression vector pTAT to construct recombinant expression vector pTAT-mlrp, The His-TAT-mLRP fusion protein was expressed in E. coil BL21 （ DE3 ） and was used to immunize the rabbits to get rabbit anti-mLRP serum, The anti-serum was purified by the acetone precipitation method. The specificity of the rabbit anti-mLRP serum was determined by Western blot, RESULTS： The predicted length of mlrp cDNA was 1 905 bp, The enconding region of the cloned mlrp cDNA, 1554 bp, was inserted into pTAT, The His-TAT-mLRP fusion protein was expressed successfully in E. coli. The rabbit anti-mLRP serum was prepared by immunizing the rabbit with mLRP protein, CONCLUSION： The successful expression of mLRP and the preparation of rabbit anti-mLRP serum lays the a foundation for further study of the function of mLRP.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2006年第3期371-373,376,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(No.30400413)
关键词
脂多糖应答蛋白
原核表达
多克隆抗体
lipopolysaccharide response protein
prokaryotic expression
ployclonal antibody
