[Objective] To screen the key catalytic sites that affect the reverse transcription function of intron-encoded protein (IEP) from Ll.LtrB, and to obtain the IEP mutant without reverse transcription activity. [Methods] The key catalytic sites affecting the reverse transcription of IEP were screened by sequence alignment and homology modeling methods. Then, the screened key catalytic sites were subjected to site-directed mutagenesis. The mutated IEP was combined with the Targetron vector to construct the mutated Targetron targeting system without reverse transcription function. Finally, the function of mutant IEP was verified by using the lacZ gene in Escherichia coli.[Results] The sites C164 and G214 of IEP were screened and mutated, completely inactivated the "retrohoming" function in vivo. [Conclusion] The IEP mutants of Ll.LtrB obtained in our study laid a solid foundation for further research on the structure and mechanism of group II intron.