莱茵衣藻TLP2多克隆抗体制备及其突变体鉴定  

Polyclonal Antibody Preparation and Mutant Identification of Chlamydomonas reinhardtii TLP2

作　　者：刘雁霞 陶冶[1,2] 张媛媛[1,2] 鲍冬雪 樊振川[1,2,3] 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)

机构地区：[1]天津科技大学食品科学与工程学院,天津300457 [2]天津科技大学大健康生物技术国家国际科技合作基地,天津300457 [3]天津科技大学食品营养与安全国家重点实验室,天津300457

出　　处：《食品与生物技术学报》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)。

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.

关 键 词：莱茵衣藻 TLP2 多克隆抗体 蛋白质纯化 

分 类 号：Q78[生物学—分子生物学]