摘要
目的探讨制备泡沫细胞相关基因4多克隆抗体的方法。方法将从人胎肝文库经聚合酶链反应扩增获得的泡沫细胞相关基因4基因全长cDNA序列,通过生物信息学分析,预测泡沫细胞相关基因4编码氨基酸序列的二级结构、抗原决定簇、功能结构域,并进行了多序列比对,并根据蛋白质的亲疏水性、二级结构、偶联难度及实验难度等,确定泡沫细胞相关基因4抗原13肽PKLVKEEVFWRNY,采用固相多肽合成法合成该抗原片段,偶联后经过基础免疫,6次加强免疫,经追踪检测,于第四次免疫后10～14天颈动脉放血,分离血清,冷冻抽干,-20℃保存而制备了泡沫细胞相关基因4兔抗人多克隆抗体。结果用固相多肽合成法成功制备了泡沫细胞相关基因4兔抗人多克隆抗体,该抗体经间接酶联免疫吸附测定法检测其效价为1∶16000,Westernblot检测可见40kDa处有目的带,用免疫组织化学方法从HepG2细胞中检测到泡沫细胞相关基因4蛋白表达,证实该抗体具有较好的反应性和特异性。结论固相多肽合成法省时、省力、制备的抗体效价高;泡沫细胞相关基因4兔抗人多克隆抗体的制备,为进一步研究泡沫细胞相关基因4蛋白在动脉粥样硬化中的功能作用奠定了基础。
Aim To explore the preparation of the polyclonal antibody of a novel Homo sapiens synapse associated protein （ FRCA）. Methods FRCA full-length sequence was obtained by PCR from human fetal fiver library, by bioinformatics to detect the second structure of amino acids encoded by FRG4 and its epitope and motifs, according to the hydrophifity, second structure, coupling and experiment difficulty ete, the peptide with 13 amino acid （PKLVKEEVFWRNY） was selected and synthesized by Sofid-phase Peptide Synthesis （SSPS） method. After eoupfing, the peptide was immunized to rabbits through the basic and 6 fortified-immunities. 10 to 14 days later just after the fourth fortified-immunity, serum, separated from blood exsanguinated from carotid,was lyophihzed and then stored at -20℃. Results Analyzed by ELISA and Western blot,the antibody dilution was 1：16 000 and the blot was showed at the location of 40 kDa,the FRG4 protein was detected in cytoplasm of HepG2 cells by immunohistory. All these proved that the antibody has a good reaction and speciality. Experiments showed that the SSPS method was convenient to control the synthesis of peptides, spare time and labours, also obtain a good dilution. Conclusion A novel Homo sapiens synapse associated protein （FRG4） antibody was synthesized successfully, and worked as a foundation to the further study of FRG4 protein function playing in cardiovascular disease.
出处
《中国动脉硬化杂志》
CAS
CSCD
2005年第3期263-266,共4页
Chinese Journal of Arteriosclerosis
基金
国家自然科学基金(30200103)
湖南省自然科学基金(02JJY4011)资助
