摘要
目的以重组人碱性成纤维生长因子为免疫原,制备高效价抗hbFGF抗血清。方法通过PCR方法改造5’编码区的12个密码子,构建hbFGF原核表达载体并在大肠杆菌(E.coli)中表达,以纯化的hbFGF免疫新西兰兔,制备高效价抗血清,用于重组hbFGF的免疫印迹分析。结果经过改造的hbFGF基因在E.coli中获得较高水平表达。从可溶性部分纯化得到纯度95%以上的重组hbFGF,以该重组蛋白免疫兔子,在二次加强后以间接ELISA检测抗血清效价可达1∶512000。免疫印迹分析显示该抗血清与E.coli中表达的重组hbFGF和标准hbFGF均有特异性反应,但与某些细菌蛋白存在弱交叉反应,经E.coli菌体蛋白吸附的抗血清,与菌体蛋白的弱交叉反应消失。结论以纯化的重组hbFGF为免疫原制备了高效价的特异性抗血清,经菌体蛋白吸附可消除存在的交叉反应性。
Objective To prepare anti-human basic fibroblast growth factor (hbFGF) serum using recombinant hbFGF as an immunogen. Methods The prokaryotic expression vector for hbFGF was constructed using 5' terminal 12 codons modified by PCR, and was expressed in Escherichia coli (E. coli ). The high titer anti-hbFGF serum was prepared by immunizing New Zealand rabbits with purified hbFGF, and then analyzed by immunoblotting assay. Results The modified gene of hbFGF was highly expressed in E. coli. The recombinant hbFGF was purified (>95%) from the soluble part and this recombinant protein was used to immunize rabbits. The titer of the antiserum after the second booster immunization was 1∶512 000 as determined by indirect ELISA. Immunoblotting analysis showed that this antiserum had specific reaction with both standard hbFGF and the recombinant hbFGF expressed in E. coli , and even had weak cross-reactivity with some bacterial proteins. This weak cross-reactivity was abolished after treating the antiserum with bacterial lysates. Conclusion High titer antiserum against hbFGF is produced using purified recombinant hbFGF as the immunogen and the weak cross-reactivity of the anti-serum can eliminated by bacterial lysate absorption.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2005年第3期186-189,共4页
Immunological Journal
基金
国家"十五"重大专项基金(2002AA2Z3344)
广东省"十五"重大专项基金(2001A1090208)资助项目
