Characterization Of Kunitz-type Serine Protease Inhibitor Hc-spi-i2 Gene Of Haemonchus Contortus

Haemonchus contortus is a gastrointestinal nematode that mainly parasitizes in the abomasum of ruminants such as cattle,sheep,and camels.After infection,it causes weakness,anemia and even death to the host animals,which seriously affects the development of animal husbandry and has great economic significance in the world.Early development of anti-nematode drugs has allowed H.contortus to be controlled to a certain extent,but with the irrational use of drugs,the gradual increase of drug-resistant strains has led to a decline in the control effects.The process that nematodes invade hosts is an important stage in the life history.Finding the key drug targets which are involved in the process of nematodes invasion and developing new antinematode drugs are particularly important for prevention and treatment.Kunitz-type serine protease inhibitors can resist the degradation of host serine protease during invasion,evade immune response,and facilitate the parasitism in the host.Therefore,screening the H.contortus serine protease inhibitor-related genes and exploring the mechanism of action and related functions will provide new ideas for the prevention and treatment of the disease.In this study,the protease inhibitor gene whose expression levels increased during invasion was selected from H.contortus transcriptome database.The CDS sequence was amplified from sheep c DNA and named Hc-spi-i2,its gene structure,functional domains and molecular evolution were predicted and analyzed.The expression of Hc-SPI-I2 was observed in female adult H.contortus by preparing polyclonal antibodies and also observed location in HEK 293 T cells.The sheep GNB1 protein that interacts with Hc-SPI-I2 was screened by using yeast two-hybrid that interacts with Hc-SPII2 in the sheep blood c DNA library,and verify the interaction relationship through Coimmunoprecipitation and co-localization experiments.The effects of Hc-SPI-I2,GNB1 and the two on NLRP3 inflammasome were explored.1.Hc-spi-i2 gene characteristic analysis and expression characteristic analysis.Obtain the reference sequence of the selected gene(Gene ID: HCON＿00133150)through Worm Base Parasite website,and it can be found that it contains 12 introns and 13 exons by analyzing.The CDS sequence of Hc-spi-i2 was amplified by using H.contortus ZJ strain c DNA as a template,with a length of 3108 bp.Analysis of the amino acid sequence showed that there are 1 signal peptide and 2 Kunitz-type serine protease inhibitor domains,and the sequence of the functional domains named Hc-spi-486i2 was amplified,with a length of 486 bp for subsequent experimental research.Through searching for homologous sequences of Kunitz-type domains in parasites,all sequences were analyzed by MEGA X,and drawing the evolutionary tree,we can see that Kunitz-type functional domains are relatively conservative.In order to study the expression characteristics of Hc-SPI-I2,q RT-PCR was used to detect the transcription level of Hc-spi-i2 in various stages of H.contortus.It was found that the transcription level was significantly increased from L3 to L4,which verified the basis for screening the proteome database.The prokaryotic expression system was used to induce Hc-SPI-486I2 protein expressing.After purification and identification,BALB/c mice were immunized to prepare polyclonal antibodies which were verified by Western Blot.Polyclonal antibodies were used to detect the localization of Hc-SPI-486I2 protein in females with the highest transcription level.Hc-SPI-486I2 was widely distributed in the intestines,gonads and subcutaneous tissues of the worms,and there were spot-like aggregations.In order to comprehensively analyze the expression in different systems,EGFP flurescent tag plasmid was constructed and transfected HEK 293 T cells to observe the localization.It showed that Hc-SPI-I2 was mainly expressed in the cytoplasm in cells.The researches in this chapter lay the foundation and provide direction for subsequent experimental research through the analysis of the characteristics of Hc-spi-i2 gene and the expression characteristics of Hc-SPI-I2 protein.2.Screening and verification of interaction protein with Hc-SPI-I2Kunitz-type serine protease inhibitors are significant in defending against host digestive tract serine proteases,and it is of great significance to explore the interaction between Hc-SPI-I2 protein and host proteins during invasion.In order to explore the unknown interacting proteins,the yeast Hc-SPI-I2 bait protein pellet was constructed,and the yeast two-hybrid system was used to screen the interacting proteins in the sheep blood c DNA library.After analyzing and comparing the screening results,9 interacting protein sequences was obtained.GNB1 protein sequence were selected and analyzed through NCBI website.The GNB1 gene was amplified and constructed to p GBKT7 yeast plasmid for mutual verification.The two eukaryotic expression plasmids with different fluorescent tags were constructed.The they were transfected into HEK 293 T cells and it was found that there was a co-localization situation,which provided support for the mutual interaction between the two proteins.Plasmids with Flag and HA antibody tags was constructed,and the Co-immunoprecipitation experiments were performed to verify that the interaction between Hc-SPI-I2 and GNB1,and there was no non-specific binding.The screening and verification of interaction proteins between H.contortus and host indicate that Hc-SPI-I2 does play a role in the process of invasion.This chapter provided a research basis for subsequent functional studies.3.The effect of protein interaction on NLRP3 inflammasomeInflammation occurs in the process of Haemonchus contortus invading the host.The inflammasome is a multimeric protein complex formed in cells,which participates in the hosts’ defense mechanism against pathogens.In HEK 293 T cells that naturally lack NLRP3 and ASC,an in vitro model of NLRP3 inflammasome was reconstructed for subsequent investigation.THP-1 cells c DNA stimulated by LPS and ATP was used as a template,four genes required to construct NLRP3 inflammasome were amplified,and obtained the correct sequences of Caspase-1,NLRP3,ASC and IL-1β.Through comparing and analyzing the functional domains of 4 human genes and sheep genes,it can be seen that the NLRP3 inflammasome is relatively conserved between the two species.The p Lenti CMV tag plasmids were constructed and were co-transfected into HEK293 T cells.On the basis of Western Blot verification and expression,the IL-1β transcription level was significantly up-regulated by q RT-PCR,showing that the NLRP3 inflammasome was successfully reconstructed.In order to explore the effects of Hc-SPI-I2 and GNB1 proteins on NLRP3 inflammasomes,the above two plasmids were transfected separately and co-transfected in the reconstitution system,and it was found that both NLRP3 inflammasome could be significantly inhibited,and the interaction between the two can promote this inhibition.In conclusion,this study amplified the Hc-spi-i2 gene whose transcription level was significantly increased during the invasion stage of Haemonchus contortus,analyzed its gene structure and Kunitz-type functional domain in bioinformatics.Through prokaryotic expression to prepare polyclonal antibodies and eukaryotic cell transfection to clarify the expression characteristics of Hc-SPI-I2 protein in different systems.There are 9 proteins that interact with Hc-SPI-I2 were screened through yeast two-hybrid,and GNB1 was selected to verify the interaction relationship in different ways.The inhibitory effects of Hc-SPI-I2 and GNB1 on NLRP3 inflammasome were explored by reconstructing the NLRP3 inflammasome.The experimental results in this paper lay a foundation for the interaction and inflammatory response between H.contortus and the host during the invasion stage,and also provide a new direction for the prevention and treatment of Haemonchus contortus.