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

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（isopropyl β-D-thiogalactoside，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∶51 200，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。

Abstract:

[Objective] This study aimed to prepare rabbit anti-Chlamydomonas reinhardtii (C. reinhardtii) Tubby-like 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 isopropyl β-D-thiogalactoside (IPTG). New Zealand white rabbits were immunized with the purified 6×His-TLP2. [Result] The antiserum titer determined by indirect ELISA method reached 1:51 200. 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.