Differential Expression Of TgMIC1 In Different Virulent Strains Of Toxoplasma Gondii Chinese1 Genotype

Background:Toxoplasma is a kind of obligate intracellular parasite.It can infect humans and all warm-blooded animals.It is an important zoonotic pathogen,which has a great impact on human health and animal husbandry production.T.gondii is the only specie in Toxoplasma,however,researches on the genetic structure of its population shows that T.gondii in the world is genetic diversity.The major genotypes that are prevalent in North America and Europe are the classic Type1,Type2 and Type3,which show strong,medium,and weak virulence characteristics in mice.The genotype of T.gondii in China is different from the clonal lineage of other continents in the world.The dominant genotype is Chinese 1(ToxoDB#9),including strong virulene strain(TgCtwh3)and weak virulene strain(TgCtwh6).Previous studies on T.gondii virulence factors and their polymorphisms have focused on the classic Typel,Type2,and Type3,and little was known about Chinese1 that prevalent in China.We previously analyzed the transcriptomes and genomes of the two representative T.gondii strains in China,and found that many proved virulene effector molecules(ROP18,ROP5,ROP16,GRA15)showed no significant difference in gene st-ructure and transcription level between TgCtwh3 and TgCtwh6,but tra-nscriptome sequencing analysis found that the transcription level of MIC1 was significantly increased in the virulent strain TgCtwh3.MIC1 is a micronemes(MIC)protein that forms a complex with two other MICs,MIC4 and MIC6(MIC1-MIC4-MIC6),and plays an important role in the process of Toxoplasma invasion into host cells.In addition,the latest research also found that MIC1 can bind to TLR2 and TLR4 receptors of mouse dendritic cells and macrophages,cause cytokine storms and host death,and play important immune regulatory functions.Based on this,we speculate that the differential expression of MIC1 in TgCtwh3 and TgCtwh6 may be related to the difference in virulence between the twoObjective:To study the differential expression of MIC1 in TgCtWh3(strong virulence)and TgCtWh6(weak virulence)of T.gondii dominant genotype Chinese1 in China,and to explore the relationship between its differential expression and Chinese1 genotype virulenceMethods:(1)Differences in transcription levels of MIC1 between TgCtwh3 and TgCtwh6:TgCtwh3 and TgCtwh6 strains were passaged intraperitoneally,and 1 × 107 tachyzoites reached were collected.RNA was extracted with TRIzol and chloroform,and reverse-transcribed into cDNA.Real-time fluorescence quantitative PCR(qRT-PCR)technology wasconducted to determine the mRNA lever of MIC 1,using ?-tublin as the internal reference gene,and 2'-??CT method was used to calculate the relative transcription levels of target genes such as MIC1(2)Preparation and identification of MIC1 polyclonal antibody:The Optigene TM codon optimization analysis platform was used to optimize the coding sequence of the MIC1 functional region,the optimized coding gene was synthesize,and then clonen into the pET30a prokaryotic expression vector to construct pET30a-MIC1 recombinant plasmid.The recombinant plasmid was transformed into Escherichia coli BL21(DE3),and the recombinant protein was induced and IPTG.After purification by His affinity chromatography,His monoclonal antibody was used as the primary antibody for Western blot identification.New Zealand white rabbits were immunized with the purified fusion protein to prepare polyclonal antibodies.After purification,the titers and specificities were analyzed by ELISA and Western blot,respectively(3)Differences in the expression levels of MIC 1 between TgCtwh3 and TgCtwh6:1 ×107 tachyzoites of TgCtwh3 and TgCtwh6 were collected.After protein extraction,the protein level was detected by Western blot.Toxoplasma actin was used as the internal reference.To calculate the relative expression levels.(4)Effect of differential expression of MIC 1 on the invasion ability of Chinesel T.gondii:1×106 HFF cells were plated in 12-well plates.Take freshly lysed 3x106 tachyzoites were added to HFF and Three groupes were designed:TgCtwh3,TgCtwh6,TgCtwh3+Anti MIC1,Two hours after infection,cells were fixed,rabbit-derived anti-Toxoplasma GAP45 was used as primary antibody,and goat anti-rabbit Alexa Fluor 488 was used as a fluorescent secondary antibody to mark extracellular invasion T.gondii,and then GAP45 was used as the primary antibody,and goat anti-rabbit Alexa Fluor 594 was used as the secondary antibody to mark intracellular and extracellular tachyzoites.Randomly selected 10 fields of view taken under a fluorescence microscope were used to calculate the numbers of intracellular and extracellular tachyzoites.The proportions of intracellular tachyzoites to total tachyzoites were calculated and represent-ed the invasion efficiency.Results:(1)The qRT-PCR results showed that the transcription level of the MIC1 gene in the TgCtwh3 strain was 3.05 times that in the TgCtwh6 strain.(2)Through the optimization of MIC 1 by the Optigene TM codon optimization analysis platform,we obtained the MIC1 coding gene with better gene expression efficiency,successfully constructed the pET30a-MIC1 recombinant plasmid,and obtained highly efficient expression.The relative molecular mass of the recombinant MIC1 was about 60kd.The concentration of the purified MIC1 polyclonal antibody reached 2.5 mg/ml,and the titer was 1:12800 by ELISA.The MIC1 polyclonal antibody can recognize the MIC1 expressed by recombinant bacteria and the natural MIC1 protein expressed in RH,TgCtwh3 and TgCtwh6,It has no crossreaction with pET30a-MIC4,pET30a-MIC6,pET30a-ROP18,Plasmodium and Schistosoma.(3)Western blot results showed that the relative expression of MIC 1 in TgCtWh3 in TgCtwh3 was 1.67,and the relative expression of MIC1 in TgCtwh6 was 0.72,The expression of MIC1 in TgCtwh3 was 2.31 times that in TgCtwh6(4)The invasion efficiencys of three groups:TgCtwh3,TgCtwh6,TgCtwh3+ AntiMIC1 are 67%,40%,and 43%,respectivey.The invasion efficiency of TgCtwh3 is higher than that of TgCtwh6(p<0.01).MIC1 can significantly reduce the invasion efficiency(p<0.001)Conclusion:The TgCtwh3 strain has significantly higher transcription and expression levels of the MIC1 gene than that in TgCtwh6 strain.The prepared MIC1 polyclonal antibody can specifically recognize the MIC1 protein of different T.gondii strains and the recombinantly expressed MIC1 protein.The invasion efficiency of TgCtwh3 in HHF was higher than that of TgCtwh6,and the MIC1 polyclonal antibody significantly reduced the invasion of TgCtwh3.