Genome Analysis And Function Study Of The Hemolysin Gene Of Mucinivorans Hirudinis

Hirudo nipponica,one of the baszoanimals of the medicinal material leech HIRUDO,is included in the Chinese Pharmacopoeia(2020 edition)and can be used for the treatment of blood stasis and amenorrhea,stroke hemiplegia and fall injury.In recent years,due to the increasing environmental pollution,the wild resources of Hirudo nipponica are less and less,but the demand for them is increasing year by year,and the contradiction between supply and demand is very prominent.In order to make up for the shortage of natural resources of Hirudo nipponica,to protect the Hirudo nipponica germplasm resources,artificial breeding has become a necessary way.However,in the process of Hirudo nipponica,it was found to have weak digestive capacity and slow growth because of the lack of digestive enzymes in its intestine.How to solve the digestive problem of the leech has become an objective need to improve the yield of leech breeding.Studies have shown that animals’ gut commensal bacteria play an active role in digestion.Therefore,in this study,combined with the rules of intestinal microbial changes of Hirudo nipponica before and after feeding blood,we isolated Mucinivorans hirudinis from its gut,analyzed it genome,selected the hemolysin gene,and studied its function.The results of the study are as follows:(1)Comprehensive analysis of gut microbial structure and diversity in different periods before and after the blood meal was conducted using high-throughput sequencing in this study.The results showed that there were obvious differences in the intestinal microbial structure before and after feeding in Hirudo nipponica,and the intestinal microbial richness and diversity were lower after feeding a blood meal than before feeding blood.After feeding the blood meal,two dominant genera were produced in the gut of the leech,namely Mucinivorans and Aeromonas,with significantly increased relative abundance compared to that before feeding blood.(2)Intestinal microorganisms was isolated from the gut of Hirudo nipponica and molecular identified by 16 S rRNA sequencing.Based on the 16 S rRNA gene phyloevolutionary analysis,the isolates were 97.88% similar to the known Mucinivorans hirudinis strain M3.To resolve the genome of Mucinivorans hirudinis,the result showed the genome size of 3149227 bp and G + C content of 54.11%.The annotation of carbohydrate active enzyme database indicates that more number of glycoside hydrolase genes are annotated and two genes are annotated as hemolysins in this genome.The hemolysin gene numbers are M.hirudinis＿GM000585 and M.hirudinis＿GM001846.(3)Two hemolysin genes were analyzed by bioinformatics.The results show that the protein encoded by MhlyA585 has 266 amino acids,and the protein encoded by MhlyA1846 has 275 amino acids,none of which has a signal peptide.The MhlyA1846 has the same functional domain as the Bactericides hemolysin,but the MhlyA585 does not have.Based on this,the MhlyA1846 sequence was synthesized using the whole gene synthesis method,and the double-enzyme digestion method was used to obtain the MhlyA1846 recombinant plasmid,then construct the prokaryotic expression system.After the expression and the purification,the result showed that the recombinant protein was obtained in the supernatant by SDS-PAGE,and that the recombinant protein size was about 36.0 k Da,the recombinant protein matches the theoretical size of the target protein.The results of hemolytic activity showed that p ET28a-MhlyA1846 recombinant protein has hemolytic activity.Through the above studies,this paper explored the rules of Hirudo nipponica intestinal microbial changes after feeding blood,and clarified whether the hemolysin from Mucinivorans hirudinis has hemolytic activity,it lays a theoretical foundation for the study of intestinal microorganisms in promoting the digestion of Hirudo nipponica and even blood-consuming animals.