摘要
为探索骨桥蛋白(OPN)调控骨代谢及与肿瘤的关系,本试验利用DNAMAN与Mega 5.02软件分析OPN蛋白系统进化关系;采用RT-PCR与分子克隆方法制备OPN蛋白表达载体;利用SDS-PAGE电泳切胶纯化、尿素浓度梯度复性获得OPN蛋白,免疫小鼠制备OPN蛋白多克隆抗体;采用ELISA法检测抗体滴度,Western blotting检测抗血清特异性。OPN序列的同源性比对分析发现,在不同进化层次动物中均存在Arg-Gly-Asp(RGD)短肽重复序列;OPN序列系统进化分析显示,OPN在动物间具有明显的进化趋势;提取小鼠肝脏RNA,OPN重组载体双酶切、DNA测序鉴定结果及蛋白表达、纯化条带大小均与预测一致;ELISA法确认OPN抗血清滴度达1∶1 600;Western blotting证实抗血清具有很好的特异性。结果表明本试验对OPN序列进行了遗传进化分析,成功克隆、表达、纯化及复性OPN蛋白,并制备、鉴定了小鼠多克隆抗体。
In order to explore the function of osteopontin （OPN） regulated bone metabolism and the relationship between OPN and tumor,DNAMAN and Mega 5.02 softwares were used to ana- lyze phylogenetic relationships of OPN protein,the OPN expression vector was prepared by RT- PCR and molecular clone; OPN was purified by SDS-PAGE gel slices,renatured by urea concen- tration gradient,and immunized mice to prepare the polyclonal antibody; The antibody titer and specificity were detected by ELISA and Western blotting, respectively. The homology comparison result of OPN sequence showed that it existed short repeat sequenc Arg-Gly-Asp （RGD） in dif- ferent animals with evolution levels. Phylogenetic tree of OPN sequence showed that OPN had ob- vious evolution trend among animals. RNA was extracted, OPN recombinant vector had been di- gested and sequenced to confirm the correct construction,and strip lengths of OPN expression and purification were agreed with the prediction. The OPN antiserum titer was 1 ：1 600 detected by ELISA,and Western blotting proved that the antiserum had good specificity. We had successfully analyzed genetic evolution relationship of OPN sequence, gotten OPN expression vector, purified and renatured OPN,and prepared and identified the polyclonal antibody against OPN.
出处
《中国畜牧兽医》
CAS
北大核心
2015年第5期1069-1075,共7页
China Animal Husbandry & Veterinary Medicine
基金
陕西省教育厅科学研究计划项目(2013JK0723)
陕西理工学院人才启动项目(SLGQD13-15)
