Polyclonal Antibody Preparation and Mutant Identification of Chlamydomonas reinhardtii TLP2

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.