大鼠神经球蛋白的原核表达及多克隆抗体制备

目的 克隆Wistar大鼠神经球蛋白(NGB)基因,通过原核表达制备其多克隆抗体,为进一步研究其功能奠定基础.方法 从雄性Wistar大鼠脑组织中提取总RNA,经逆转录PCR得到cDNA,测序证明该基因序列正确后,亚克隆人pET-28a( )载体中,通过双酶切和测序确定序列和阅读框正确.转化大肠杆菌BL21菌株,获得可溶性表达产物,经鉴定、纯化后免疫新西兰兔,分离血清,ELISA法测定抗体效价.结果 神经球蛋白多克隆抗体效价达1:100 000,此多抗可以与293细胞内表达的重组蛋白发生很好的抗原抗体反应.结论 成功制备了兔抗NGB的多抗,为后续研究提供了重要的实验材料.

Prokaryotic expression and polyclonal antibody preparation of rat neuroglobin gene

Objective To clone rat NGB gene and prepare its polyclonal antibody in order to investigate the function of neuroglobin (NGB) Methods The total RNA was extracted from Wistar rat brain and the full length cDNA encoding NGB was obtained by RT-PCR Confirmed by sequencing analysis,it was inserted in the prokaryotic expression vector pET-28a( ) pET-28a( )-NGB was expressed in Ecoli BL21 after its sequence and reading frame were confirmed by two restriction endonucleases and sequencing The protein of pET-28a( )-NGB was used to immunize New Zealand rabbits for preparing polyclonal antibody after identification and purification The antibody titer was tested by ELISA method Results The titer of polyclonal antibody was 100 000 Immunoblot analysis showed the polyclonal antibody can recognize fusion protein expressed in 293 cells Conclusion The polyclonal antibody of NGB is prepared and can be used in further study