| Background and purpose of research:Toxoplasma gondii is an important obligate parasitic protozoon in the apex gate and sporozoa.Cats are the primary host of Toxoplasma gondii.Humans and other animals act as intermediate hosts.Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii infection.Epidemiological survey shows that in Britain and the United States,the prevalence rate of adult infection is generally about 16-40%,and some are70%.The prevalence rate of adult infection in continental and Latin American surveys is 50-80%,and the French are up to 90%.Toxoplasma gondii,there are 25% to 50% infection people in the world,the positive rate of infection in China is 5% to 20%,and in some areas is more than30%.It is estimated that 25% to 50% of the world’s Toxoplasma infection is due to serious opportunistic parasites.Toxoplasma infection is becoming a worldwide and serious public health problem with the increasing number of tumor patients and pets raising.Currently,the main drugs for controlling toxoplasmosis include sulfadiazine,pyrimethamine,aeetylspiramycin,spiramycin,etc.In recent years,these drugs have low radical cure rate,long course of treatment,large toxic and side effects,and only effective for tachygonite of Toxoplasma gondii,they have no effect on the retardedcolonies,and they are easy to relapse after stopping the drug.These shortcomings have caused a lot of controversy.Therefore,finding new drug targets and developing new anti-Toxoplasma drugs has become the focus of current research on Toxoplasma infection.Most eukaryotes maintain their homeostasis redox dynamic balance by relying mainly on two sets of independent antioxidant systems,one is the Trx system,and the other is the GSH system.Parasites also need to maintain redox balance in the host to ensure parasitic parasites.The study found that parasitic worms have a nicotinamide adenine dinucleotide(coenzyme)phosphoric acid dependent thioredoxin-glutathione reductase(TGR)redox control system.Thioredoxin glutathione reductase(TGR)has the functions of these two enzymes,thioredoxin reductase(TrxR)and glutathione reductase(GR).At present,researchers have used thioredoxin reductase as an important target for the development of anti-infective agents in worms.Most parasitic protozoa contain thioredoxin(Trx)system,which has thioredoxin reductase.Compared with mammalian thioredoxin reductase,they have different redox centers.This study suggests that the antioxidant system,thioredoxin reductase,which is dependent on Toxoplasma gondii,is necessary for the growth of Toxoplasma gondii,and objective to explore the feasibility of using Toxoplasma gondii TrxR as a drug target.Method:1.The TrxR gene and protein of Toxoplasma gondii were analyzed by bioinformatics.2.RNAi interfered with Toxoplasma gondii thioredoxin reductase.3.The effect of RNAi interference on the proliferation of Toxoplasma gondii Tachyzoites was detected under light microscope.4.The relative expression of thioredoxin reductase is detected by the fluorescence quantitative PCR method.5.Measure the activity of thioredoxin reductase after RNAi interference.Result:1.The TrxR-1 gene of Toxoplasma gondii and other 7 species had no significant codon bias but significant difference and the codon preference of Toxoplasma gondii is closest to humans.Thioredoxin reductase protein of Toxoplasma gondii is also different from other species.It indicated that thioredoxin reductase of Toxoplasma gondii has certain specificity.2.Transfer of Toxoplasma 24 h with specific siRNA,the results of the3 experimental groups and the negative control group are respectively0.54±0.09,0.33±0.04,0.21±0.03,0.93±0.08.The negative control group is significantly higher than the experimental group,the differences are statistically significant(P<0.005).3.Quantitative expression of thioredoxin reductase in Toxoplasma gondii after 24 h was detected by real-time PCR.The results of the 3experimental groups and the negative control group are respectively0.54±0.09,0.33±0.04,0.21±0.03,0.93±0.08.The negative control group is significantly higher than the experimental group,the differences are statistically significant(P<0.005).4.The results of the 3 experimental groups,the negative control group and the control group are respectively 0.000441±0.000098,0.0002205±0.000084,0.000147±0.000049,0.001764±0.000147.There is a significant difference between the experimental group and the negative control group,and the differences are statistically significant(P<0.005).Conclusion:1.Toxoplasma thioredoxin reductase is typically different from human and the other animal related enzyme.2.RNAi interferes with Toxoplasma gondii thioredoxin reductase can effectively inhibit the proliferation of Toxoplasma gondii in cells and reduce the expression of thioredoxin reductase,which can be used as a drug target site. |
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