摘要
【目的】制备兔抗莱茵衣藻(Chlamydomonas reinhardtii,C.reinhardtii)类Tubby家族蛋白2(TLP2)多克隆抗体来鉴定莱茵衣藻tlp2突变体,研究莱茵衣藻TLP2的生物学功能。【方法】通过聚合酶链式反应(PCR)获得tlp2目的基因片段,构建原核表达载体pET-28a(+)-tlp2、pMAL-c2x-tlp2。将2种重组质粒通过化学转化的方法转至大肠杆菌(Escherichia coli,E.coli)BL21(DE3),经异丙基-β-D-硫代半乳糖苷(isopropylβ-Dthiogalactoside,IPTG)诱导表达融合蛋白6×His-TLP2及MBP-TLP2。将纯化后的6×His-TLP2融合蛋白免疫新西兰大白兔来获得抗血清。【结果】通过间接ELISA法测定抗血清效价达1∶51200,对抗血清进行protein A和抗原抗体纯化,在1∶500的稀释条件下,蛋白质免疫印迹表明TLP2抗体能够特异性识别莱茵衣藻TLP2。【结论】作者制备出了高特异性、高灵敏度的兔抗莱茵衣藻TLP2多克隆抗体,并鉴定出一株tlp2突变体,可用于后续莱茵衣藻TLP2在纤毛生成和纤毛信号转导中的功能研究。
[Objective]This study aimed to prepare rabbit anti-Chlamydomonas reinhardtii(C.reinhardtii)Tubbylike protein 2(TLP2)polyclonal antibodies for the identification of C.reinhardtii tlp2 mutants and to investigate the biological functions of TLP2 of C.reinhardtii.[Method]The tlp2 gene fragment was obtained by polymerase chain reaction(PCR),and the prokaryotic expression vectors pET-28a(+)-tlp2 and pMAL-c2x-tlp2 were constructed.The two recombinant plasmids were transferred to Escherichia coli(E.coli)BL21(DE3)by chemical transformation,and the expression of the fusion proteins 6×His-TLP2 and MBP-TLP2 was induced by isopropylβ-Dthiogalactoside(IPTG).New Zealand white rabbits were immunized with the purified 6×His-TLP2.[Result]The antiserum titer determined by indirect ELISA method reached 1:51200.The antiserum was purified by protein A and antigen affinity chromatography.The TLP2 antibodies specifically recognized TLP2 of C.reinhardtii at a dilution factor of 1:500.[Conclusion]Highly specific and sensitive rabbit polyclonal antibodies against TLP2 of C.reinhardtii were prepared,and a tlp2 mutant was identified.The mutant can be used for subsequent study on the role of TLP2 of C.reinhardtii of in cilium formation and cilium signal transduction.
作者
刘雁霞
陶冶
张媛媛
鲍冬雪
樊振川
LIU Yanxia;TAO Ye;ZHANG Yuanyuan;BAO Dongxue;FAN Zhenchuan(College of Food Science and Engineering,Tianjin University of Science and Technology,Tianjin 300457,China;National International Science and Technology Cooperation Base for Health Biotechnology,Tianjin University of Science and Technology,Tianjin 300457,China;State Key Laboratory of Food Nutrition and Safety,Tianjin University of Science and Technology,Tianjin 300457,China)
出处
《食品与生物技术学报》
北大核心
2025年第1期99-106,共8页
Journal of Food Science and Biotechnology
基金
国家自然科学基金面上项目(32070698)
国家自然科学基金青年基金项目(32200558)。
关键词
莱茵衣藻
TLP2
多克隆抗体
蛋白质纯化
Chlamydomonas reinhardtii
TLP2
polyclonal antibodies
protein purification
