BLT Stages

Abstract bachelorproef 2016-2017: Characterisation and protein expression optimisation of the E75 nuclear receptor in Lepeophtheirus salmonis

Lepeophtheirus salmonis (L. salmonis), commonly found as an ectoparasite on salmonids, has a negative impact on aquaculture. Restrictions on growth of the salmon farms and gaining resistance of L. salmonis when using the current treatments, lead to an increasing need of creating new and specific treatments. Therefore, knowledge about the biology and molecular biological mechanisms of the sea lice need to be obtained. The aims of this project were to define the optimal conditions on expression of the E75 nuclear receptor, to determine if E75 contains a heme group and to examine the effect of knockdown of the E75 gene on L. salmonis larvae. Different cloning constructs were made and the ligand binding domain (LBD) of E75 in pETGB1a vector was expressed in E. coli BL21 and Rosetta cells using several conditions including the presence and absence of 5 µM hemin, before analysing heme using a fluorescent method. Knockdown of E75 was performed by integrating dsRNA into the cytoplasm of nauplia I and incubating to the copepodid life stage.

Expression in BL21 cells was not efficient because of the presence of rare codons in L. salmonis. When the E75 LBD was expressed in Rosetta, overnight expression for 20 h on 18 °C showed the best results. Heme measurement showed the highest peak (1.5) when E75-LBD was expressed in the presence of hemin. Subtraction of the blanks suggest that the signal from the measurement is derived from a heme group present in E75-LBD. After the RNA interference (RNAi) experiment in L. salmonis larva, most larva were alive and showed a normal morphology. qPCR analysis did not show knockdown of the E75 gene in copepodids. No significant difference was observed since the p-values were high (0.34 and 0.71).

The optimal expression conditions of E75-LBD cloned in pETGB1a and expressed in Rosetta cells is overnight expression at 18 °C for 20 h induced with 1mM IPTG. Both the heme measurement and the RNAi experiment need to be repeated. The heme measurement only indicates the presence of a heme group in E75-LBD, but purified E75-LBD needs to be analysed in order to confirm. If the RNAi experiment fails again, other dsRNA constructs containing another part of the gene need to be made and the experiment has to be repeated.