Study On The Mechanism Of Serine Protease Inhibitory Molecule (RHS-2) In The Blood Digestion Of Female Ticks

Tick and tick-borne diseases have been the important diseases that had plagued the development of animal husbandry in many countries in the world and in China,and it is also a new issue threatening public health.It is urgent to find new sustainable control strategies.Therefore,it is very important to understand the basic physiological basis of ticks and to study the new prevention and control strategies for tick and tick-borne diseases.The fertility of ticks is extremely strong,and an engorged female has normal spawning of thousands.Therefore,control of reproduction is the key to control ticks.The digestion of ticks provides the basis for reproduction,while digestive enzymes play an important role in the digestion functions of ticks.Whether or not the Rhipicephalus haemaphysaloides midgut serine protease inhibitor RHS-2 has such a function and how it affects the function of the related digestive enzymes,its mechanism of action is still unclear.The Rhipicephalus haemaphysaloides serine protease inhibitor(RHS-2)is a digestion-associated molecule identified by the Shanghai Veterinary Research Institute and has inhibitory activity against serine proteinases.In order to further elucidate its role in the digestion of ticks,this study mainly carried out research from three aspects.One is to study whether RHS-2 has the inhibitory effect of digestive enzymes.The second is to study the location,change and distribution patterns of RHS-2,and the third is the proteomics comparative study of Tick stool after RHS-2 interference to analyze the mechanism of RHS-2 in blood digestion.1.Construction of pCold-1/RHS-2 recombinant expression plasmid,transformed into the BL21 host strain,and expression was induced with IPTG and recombinant proteins were obtained.The enzyme activity of cathepsin C,D,L,G,K and papain was analyzed.The results show that the recombinant protein has an inhibitory effect on the activity of these six commercially available proteases.The inhibition activity on cathepsin L was the highest,followed by papain inhibition efficiency,and the inhibition efficiency on cathepsin D,K,and G was weaker,while the inhibition efficiency on cathepsin C was the lowest.2.Immunohistochemistry were used to determine the expression site of RHS-2.The results showed that RHS-2 was concentrated in the outer wall cells of midgut.The midgut protein was extracted and the change of RHS-2 expression was detected by western blotting.Protein expression decreased in RHS-2 gene interference group,the third day of decline is more pronounced than the sixth day.3.Using the microinjection method,the non-sucking females of the experimental group and the control group were injected with the dsRNA of RHS2 and the dsRNA of the control Lucifrase.The ticks were then inoculated into New Zealand white rabbits and feces were collected on the 4th and 8th days of blood-sucking.The proteimic analysis of collected feces was performed using the Label free method.There are 198 tick-derived proteins and 697 rabbit-derived proteins were identified in tick feces.There were 38 different protein molecules in the fourth blood-sucking group compared with the control group,and 39 different protein molecules in the interference group compared with the control group.Among them,the differential expressed proteins of the tick-derived proteins are mainly heat shock proteins,immune protein-related subunits,and proteases.Rabbit-derived differential expressed proteins are mainly coagulation factors,glycoproteins,and some enzymes involved in digestion and synthesis such as rabbit serine/threonine proteins.Phosphatase,inosine triphosphate pyrophosphatase,S-(hydroxymethyl)glutathione dehydrogenase,and the like.The above results show that: RHS-2 is mainly located in the outer wall cells of the midgut lumen and it showed different degrees of inhibitory activity against various digestive proteases,affecting the digestion of host proteins and self proteins,and thus affecting the digestive function of ticks.These results contribute to our understanding on the digestive effect of the serpin molecule and the basic physiology of ticks.