Preliminary Study On Identification And Function Of Hyaluronidase From Haemonchus Contortus

Haemonchus contortus can cause serious parasitic diseases of cattle and sheep,parasitizing the abomasal of the host,causing the death of young animals and causing serious economic losses.At present,the prevention and control of Haemonchus contortus is still based on drug prevention,but the problem of nematode resistance is becoming more and more serious,and it is urgent to explore new prevention and control measures and intervention targets.Haemonchus contortus undergoes a free life stage and a parasitic stage.The infected larva is L3.When L3 is orally infected with the host,it reaches the abomasal about 3 days,and then drills into the abomasal mucosa and develops to L4 before drilling the mucosal establishment.Developed on the surface of the abomasal to the adult.Drilling into the stomach mucosa is an important part of the invasion process of Haemonchus contortus.Hyaluronidase can degrade the extracellular mucopolysaccharide hyaluronic acid.Hookworm-related studies have shown that it plays a role in the invasion process,and in vitro culture of H.contortus L3 larvae develops into L4 larvae can released Hyaluronidase.The preliminary research work of this laboratory showed that the adult development rate of Haemonchus contortus decreased after irradiation,and the expression of hyaluronidase gene was down-regulated.Therefore,we speculate that the expression level of hyaluronidase is related to the virulence of Haemonchus contortus.Based on this,we carried out preliminary research work on the function of hyaluronidase of Haemonchus contortus.First,according to the whole gene sequence of Haemonchus contortus hyaluronidase published in GenBank,the analysis indicated that the hyaluronidase gene encodes 511 amino acids with a theoretical molecular weight of57 kD.The signal peptide is predicted by signalP prediction.The target gene sequence was amplified by PCR,and then digested and sequenced,transformed into E.coli for prokaryotic expression and analyzed by Western blot.Immunohistochemical method was used to identify hyaluronidase in Haemonchus contortus L3 larvae;finally,using RNA interference technology,designing and synthesizing specific siRNA for hyaluronidase gene,and introducing siRNA into the infective L3 larvae of Haemonchus contortus by electroporation,using real-time PCR The transcriptional inhibitory effect of hyaluronidase gene in L3 larvae was analyzed.The hyaluronidase-specific siRNA was electrotransformed into L3 larvae for 24 h,then infected with sheep abomasal explants,and the hyaluronan gene was silenced.Theinvasion rate of abomasal tissue was observed,and the abomasal explants after invasion of L3 larvae were observed by tissue section.At the same time,hyaluronidase-specific siRNA was electrotransformed into L3 larvae for 24 hours,then sheep were infected,and sheep feces were collected regularly to check eggs.Number,after the 30 th day,the sheep were necropsy and the numbers of insects was checked.Preliminary results indicate that the results of western-bolt indicate that recombinant HC-HAase has good immunogenicity,and the results of immunohistochemistry show that HAase is mainly distributed in the head of the worm,which provides an effective basis for demonstrating that the enzyme plays an important role in invasion.The expression of hyaluronidase gene was detected by real-time fluorescent quantitative PCR after 24 hours of electroporation.The relative expression of hyaluronidase gene in the experimental group was significantly lower than that of the control group(p<0.05).The method can effectively reduce the enzyme activity unit of hyaluronidase by introducing hyaluronidase gene-specific siRNA into the L3 s worm.The results of in vitro experiments showed that the invasion rate of the larvae of Haemonchus contortus L3 s to gastric tissue explants after the hyaluronidase gene interference was 9.45%,while that of the control group was 53.62%.The results of animal experiments showed that the number of eggs in feces was 18-32 days after the larvae of L3 transgenic Haemonchus contortus larvae were infected by siRNA.The number of eggs per gram of feces in the interference group was significantly lower than that of the control group;After the day,the number of adults in the abomasal was examined.The colonization rate of the control group was 77.1%,and the experimental group was 39.7%,and there was significant difference between the two groups(P<0.01).The L3 larvae of Haemonchus contortus may secrete hyaluronic acid.The enzyme decomposes the host mucosal tissue,which facilitates the invasion of the host into the mucosa of the host,thereby promoting the establishment of the worm in the host mucosa.Hyaluronanase is a key virulence factor in the invasion of Haemonchus contortus,and its functional research is conducive to the prevention and treatment of Haemonchus contortus.