Protective Effect Of Haemonchus Contortus Matrix Metalloproteinase 12A On Goats

Haemonchus contortus is a parasitic nematode belongs to trichophyceae family.Ruminants get infected by the ingestion of the third stage of the larva.The disease symptoms included i.e.,anemia,diarrhea,weakness,and even death often occurred,and even causes huge economic losses,especially in animal husbandry practices.However,only one commercial vaccine is available.Finding more vaccine candidates would be necessary for the prevention of the disease caused by this parasite.The recombinant protein of matrix metalloproteinase 12A(MMP12A)of Haemonchus contortus has been proved to have a protective effect against the infection of Haemonchus contortus in goats.In this paper,the eukaryotic expression plasmid pVAX-MMP12A was constructed.The eukaryotic plasmid and recombinant protein were administrated in different combinations to study its immunoprotective effects on goats.In order to elaborate on the function of this gene,especially for the development of worms,the RNA interference technique was used to silence the MMP12A gene in the third stage of the Haemonchus contortus larvae and its ability to infect the goats was studied.1 Construction of recombinant plasmid pVAX-MMP12AThe BL21 strain with pET-32a-MMP12A plasmid was recovered and transformed into our laboratory.The MMP12A gene was extracted from the recombinant plasmid and digested by double enzymes.The MMP12A gene was connected to the eukaryotic expression vector pvax by DNA recombination technology,and the eukaryotic recombinant plasmid pVAX-MMP12A was obtained.The expression of MMP12A was detected by Western blot.2 Protective Study of MMP12A on GoatsTwenty-five healthy goats were randomly divided into 5 groups,5 in each group.The first group was the non-immune control group;the second group was the non-immune control group;the third group used recombinant plasmid pVAX-MMP12A for the first immunization and the second immunization;the fourth group used recombinant plasmid pVAX-MMP12A for the primary vaccination and recombinant MMP12A for the second immunization;the fifth group used recombinant MMP12A for the first immunization and recombinant plasmid pVAX-MMP12A for the second immunization.14 days after the second immunization,the goats in groups 2-5 were artificially infected with 8000 third-stage larvae of Haemonchus contortus(group 1 is a non-infected control group).Serum and stool samples were collected at different time points14 days after the second immunization,8000 third-stage larvae of Haemonchus contortus were artificially infected in group 2-5(the first group was the non-infected control group).Serum and fecal samples were collected at different time points to analyze the serum cytokines IFN-γ,TGF-β IL-4,IL-9 and IL-17,IgA and IgG,egg count,and adult population.The results of ELISA showed that the immunization of specific antibody IgA titers in the three immunization groups and the two control groups were not changed significantly during the entire test period,and all fluctuated within a small range;the immunization of the goat-specific serum antibody IgG in the three immunization groups was the first After the second titer was significantly increased,and continued to grow after the second immunization;serum cytokine test results showed that the IFN-y levels of the two control groups did not change significantly during the period before the attack,fluctuating within a reasonable range,The changes in the three immunization groups were more prominent,slightly increased after the first immunization,and increased significantly after the second immunization;the levels of IL-9 in the two control groups were in a lower fluctuation range before the attack,and the changes were not noticeable.Compared with the two control groups,the immunized group increased significantly after the first immunization and did not change much after the second immunization.Still,serum cytokine IL-4 level did not change significantly in the two control groups,and the three immunized groups were in the first immunization group compared with the two control groups.After the second rise,the increase is evident after the second immunization;the data of serum cytokines IL-17,TGF-β in each group change rules are not manifest;the difference is not significant.The results of the egg count and the number of adult insects showed that the non-immunized non-attack control group was 0,and the three immunized groups were compared with the non-immunized attack control group.The eggs of the third,fourth,and fifth groups were reduced.The rates were 68.14%,67.18%,and 72.17%,respectively,and the reduction rates of adult insects were 70.38%,69.43%,and 76.79%,respectively.The above results indicate that Matrix metalloproteinase 12A of Haemonchus contortus has excellent immune protection and potential application value.3 Effect of RNA silencing of MMP12A gene on body developmentSynthesize a pair of siRNA(S2)and a pair of irrelevant interfering RNA(SN)that have been confirmed to have sound interference effects on the MMP12A gene by our laboratory.At the same time,the control group was set up with PBS instead of siRNA under the same conditions.Take larvae(a small part of them)in each group to extract mRNA,reverse-transcribed into cDNA,and perform fluorescent quantitative PCR to detect interference effects.The results showed that the gene expression of group S2 decreased significantly.Twenty healthy goats were randomly divided into 4 groups(n=5),the first group was a non-insect control group;the second group(PBS group)was artificially infected with 8000 third-stage larvae of Haemonchus contortus treated with the above PBS;The third group(S2 group)was artificially infected with 8000 S.contortus third stage larvae treated with S2;the fourth group(SN group)was artificially infected with 8000 S.contortus third stage larvae treated with SN.After infection,the goats of the experimental group were tested and analyzed for egg count,egg hatching rate,adult insect load,and adult length at different time points.The results compared with the PBS group,the SN group had no significant difference in data;the S2 group compared with the PBS group,the number of fecal insect eggs per gram(EPG)decreased by 61.61%,the number of adult insects decreased by 56.42%,female and male The length of the insect body was shortened by 16.7%and 11.6%respectively.The hatching rate of insect eggs was reduced by 20.43%.The difference between the groups was significant.The above results indicated that the MMP12A gene plays a vital role in insects for growth and development.