Prokaryotic Expression Vector Construction Of Cas14a1 Related Recombinant Protein And Its Preliminary Application In Invitro Nucleic Acid Technology

Cas14a1 system is the smallest researched CRISPR systems after g RNA-guided endonuclease has been found in the CRISPR/Cas system,and deeply studied and widely used by researchers.Compared with other Cas-enzymes,Cas14a1 has the advantages of small protein molecular weight,simple composition,and its accessory activities can be activated after cleavage effects.It is expected to be the most effective and most widely used gene editing and pathogenic microorganism detection tools in the future.This study mainly explores the design,construction,purification and enzyme activity mechanism of Cas14a1-related recombinase.The main results are as follows:(1)Design and construction of the recombinant enzyme by cas14a1-helicase.In this study,Cas14a1 and Helicase protein were used as the research objects.Helicase,Helicase-Cas14a1 and Cas14a1-Helicase proteins were constructed prokaryotic,expressed and purified.We performed preliminary screening of protein prokaryotic expression conditions from three aspects: different induction temperature,time and inducer concentration(IPTG).The results showed that the expression of prokaryotic protein could be effectively improved.At the same time,the results showed that the Helicase,Helicase-Cas14a1 and Cas14a1-Helicase proteins with a single band were purified by optimizing different purification buffer and purification conditions,and the purity of the proteins reached 90%.(2)Preliminary application of Cas14a1 and its recombinase in gene-editing of vitro.In order to further explore cleavage activity and the molecular mechanism of Helicase-Cas14a1 and Cas14a1-Helicase,We mainly carried out PCR amplification,fluorescent probe and other molecular biological experiments.Firstly,g RNA sequences were obtained through in vitro transcription technology,then g RNA guided the recombinant protein to recognize the target DNA and activate its protein was activitied to cleave the target DNA.The g RNA guided the protein to recognize the target DNA,with protein activity was activated and target DNA was cleaved.The results of nucleic acid PAGE gel and fluorescent probe detection showed that the Helicase-Cas14a1 could cleave target DNA without g RNA,when the reaction time reached more than 40 min.The proteins of Helicase-Cas14a1 were non-specific to cleave the target DNA.The results showed that Cas14a1-Helicase protein did not cleave target DNA,after a series of controlled experiments excluding the influence of Helicase,g RNA and other factors.The non-specific cleavage activity may be caused by the fusion of two protein domains.The mechanism of enzyme reaction needs to be further studied,which also lays a certain experimental foundation for the follow-up researches on multifunctional proteins.