Construction Of The Transfecting Plasmid With The P24 Gene Of Toxoplasma Gondii Knocked Out And Preliminary Research Of Establishment Method On Knock-out Strain

Objective:To establish TgP24 gene-knockout strains,and Exploration on the application conditions of screening.Tg P24 gene knockout strains.Method:(1)Construction of the targeting plasmid pGB/p5-p3 for Toxoplasma gondii P24 gene(TgP24)knock out.Accoding to the genomic sequence of TgP24,four oligonudeotides primers(P1 to P4)were designed and synthesized to amplify the 5'end 2.5 kb fragment of untramlated region(UTR)and the 3'end 2.89 kb frarment of UTR of TgP24.The PCR were subdoned into pCR2.1-TOPO TA plasmid to generate the recombinant plasmid P24-5'-UTR/ TA and P24-3'UTR/TA respectively.The purified fragment of P24-5'-UTR from the digestion of P24-5'-UTR/TA with KpnⅠand BglⅡwas inserted into the KpnⅠand BglⅡsites of plasmid GRA2/Ble to generate plasmid GRA2/Ble-P24-5'UTR(designated pGB-P5);The purified P24-3'-UTR fragment from the digestion of plasmid P24-3'-UTR/TA with BamH-Ⅰand NotⅠwas inserted into the BamHⅠand NotⅠsites of plasmid pGB-P5 to generate the targeting plasmid pGB-P5/P24-3' UTR(pGB/P5-P3).(2)The condition establishment of TgP24 knock-out strain in vitro screening.Use the millipore filter(aperture 5.0um)filter and purify RH tachyzoites,Observe relatively the impact on plasmid transfected strains of electroporation parameters,such as electroporation transfection buffer, voltage,capacitance,pulse frequency,the size of electric shock Cup The impact of pest.Observe relatively the impact on the transfected strains of different host cells(HFF,3T3 and L929)in vitro on by drug screening;Observe relatively the impact on the transfected strains screened separatly in cell and combine it with low temperature treatment ecto-cells by drug screening(selectively cultured ten years in cells.collected the strains,4℃in extracellular choose five days, retropositioned to the cells cultured in the phleomycin selective medium for seven days).(3)The establishment,identification and analysis of toxoplasma P24 gene knock-out strains.Apply the optimized conditions of electroporation method for transfering RH tachyzoites;use the best conditions for screening transferred strains,then serial subcultivation,High-power microscope observe and count the formation of parasitophorous vacuole(PV),large collection and purify Toxoplasma strains,utend RT-PCR and Western-blot two methods analysis about gene and its protein expression of Toxoplasma P24 gene knock-out strains,and research on the toxicity of gene knock-out strains.Results:(1)Experimental results showed that the targeting plasmid pGB/P5-P3 was constructed by inserting 2.5 kb and 2.89 kb DNA fragment corresponding to the 5'end and the 3'end UTR of the TgP24 gene into the plasmid GRA2/Ble.The fragment P24-5'UTR was inserted into the poly linker site Kpn-Ⅰ/BglⅡupstream of the phleornycin resistance gene(ble),and the P24-3'UTR frament inserted into BamHⅠ/NotⅠsite down-stream of the same gene.(2)Microscopic examination revealed the purity of RH tachyzoites can reach 95 percent,the recovery rate of filtered strains also can reach around 70-80 percent.Optimized conditions of electroporation has improved the survival rate of RH tachyzoites(P＜0.05);We relatively observated,the cells will be used within cells,use the method of drug screening on strains in cells and low-temperature treatment ecto-cells will be more thorough;Use L929 fibroblast cell as the host cell for screening,it is easy to form a single Layer,large-volume individual,many passage frequency and slow growth,and the most strong resistant to drug.(3)High-power microscope observe and count,after about 12hr,the number of the transfected strains PV are 8/hp(per high power microscope),significantly lower than the number of the RH tachyzoites PV are 19/hp(P＜0.05);Then large collection and purification of P24 gene knock-out strains,RT-PCR,Western-blot showed that the gene and protein expression product of strains screened in L929 cells were significantly lower the groups of strains screened in HFF ang NIH-3T3; animal toxicity test results showed that mean survival time of mice attacked strains screened in L929 cells is longest.Conclusion:(1)The construction of P24 gene knock-out plasmid pGB/P5-P3 was successfully achieved,and amplified fragment at both ends of the target gene is longer,it will benefit to improve the homologous recombination efficiency.(2)Ascertain the application conditions of Toxoplasma electroporation technology,and the best screening conditions of TgP24 knockout plasmid transfected into RH strains cultured in different mammalian cell lines(use drug screening in cells and low-temperature treatment ecto-cells,drug selective concentration within 5.0μg/ml-7.5μg/ ml phleomycin,and L929 fibroblast cell have long screening time and good screening effect as selective host cell)(3)Preliminary obtaining the toxoplasma P24 gene knock-out strains.It has laid a favourable foundation for further research on the biological characteristics of TgP24 knock-out strains.