抗AFP重链可变区单域抗体融合蛋白的构建、表达及初步鉴定
Construction,expression and identification of anti-human AFP V_H single domain antibody fusion protein gene
作者：方丽;王振达;张平;赵宗蓉;章崇杰;
Author：
收稿日期： 年卷（期）页码：2007,(03):-251-254
期刊名称：华西药学杂志
Journal Name：WEST CHINA JOURNAL OF PHARMACEUTICAL SCIENCES
关键字：甲胎蛋白;单域抗体;克隆;表达
Key words：
基金项目：四川大学科技创新基金资助项目(No.2004CF09)
中文摘要
目的构建抗甲胎蛋白(AFP)重链可变区(VH)单域抗体融合蛋白基因在大肠杆菌中的表达,并初步鉴定表达产物的活性。方法从已构建的抗AFP单链抗体(ScFv)的载体中,经PCR扩增出VH单域抗体基因,再克隆到融合蛋白表达载体pET32a(+)中进行表达;用SDS-PAGE及Western-blot鉴定表达产物;并通过竞争抑制ELISA分析TrxA-VH融合蛋白的结合活性;细胞免疫组化染色分析其内化及结合情况。结果VH单域抗体基因全长339 bp,将其克隆到pET32a(+)中,转化大肠杆菌可获得高效表达,Western-blot证实在相应分子质量处,有TrxA-VH融合蛋白的显色条带。经初步纯化和复性后,获得抗AFP的VH单域抗体融合蛋白。竞争抑制ELISA及细胞免疫组化证明,表达产物具有与AFP特异结合的活性。结论成功构建了抗AFP的V单域抗体融合蛋白,为临床的应用研究奠定了基础。
参考文献
[1]Colli A,Fraquelli M,Conte D.Alpha-fetoprotein and hepato-cellular carcinoma[J].Am J Gastroenterol,2006,101(8):1939-1941.
[2]LI MS,LI PF,HE SP,et al.Promoting molecular mechanism ofalpha-fetoprotein on the growth of human hepatoma Bel 7402line cells[J].World Gastrunal,2002,8(3):467-472.
[3]Mizejewski GJ.Alpha-fetoprotein structure and function:rel-evance to isoforms,epitopes and conformational variants[J].ExpBiol Med,2001,226(5):377-408.
[4]Smyth MI,Tekeda K,Hayakawa Y,et al.Nature’s TRAIL?apath to cancer immunotherapy[J].Immunity,2003,18(1):1-6.
[5]LIUGZ,FENG J,ZHOUP,et al.Cloning,expression and radio-imm-unoimaging of single domain(VH)gene of anti-human o-varian carcinoma McAb[J].Chin J Bioch Molec Biol,2001,17(3):350-355.
[6]Abulrob,Abedelnasser,Sprong,et al.The blood-brain barriertransmigrating single domain antibody:mechanisms of transportand antigenic epitopes in human brain endothelial cells[J].JNeurochem,2005,95(4):1201-1214.
[7]萨姆布鲁克J,拉塞尔DW.分子克隆实验指南[M].第3版.北京:科学出版社,2002.1256-1260.
[8]张平,蔡美英,赵宗蓉,等.抗人AFP单链抗体基因的构建和在大肠杆菌中的表达[J].四川大学学报(医学版),2005,36(1):20-23.
[9]Schwartz JM,Ham JM.Treatment of hepatocellular carcinoma[J].Curr Treat Options Gastroenterol,2003,6(6):465-472.
[10]Davies J,Riechmann L.A ffinity improvement of single antibodyVHdomains:residues in all three hypervariable regions affect an-tigen binding[J].Immunotechnology,1996,2(3):169-172.
[11]Dumoulin M,Conrath K,Van Meirhaeghe A,et al.Single-do-main antibody fragments with high conformational stability[J].Protein Sci,2002,11(3):500-515.
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