Prokaryotic Expression Of Profilin Protein Of Toxoplasma Gondii And The Effect Of Its Antibody On The Proliferation Of Tachyzoites

Objective:1.To establish prokaryotic expression system of profilin protein of Toxoplasma gondii?TgPRF?.2.To prepare rabbit anti-TgPRF polyclonal antibody and detect its toxicity to cells and tachyzoites.3.The in vitro proliferation model of T.gondii tachyzoites?RH-green fluorescent protein,RH-GFP?was established by mouse embryonic fibroblasts?BALB/C-3T3?.To evaluate the inhibitory effect of TgPRF antibody on the proliferation of tachyzoites of Toxoplasma gondii in vitro and its effect on the growth and apoptosis of infected cells,so as to provide theoretical support and experimental data for the protective study of TgPRF vaccine.Methods:T.gondii RH tachyzoites were obtained by serial intraperitoneal passaging in BALB/c mice.Total RNA of T.gondii tachyzoites was extracted using Trizol Reagent and was reverse-transcribed into cDNA according to manufacture's instruction.Specific primers were designed to amplify TgPRF gene from the cDNA template.PCR products and pET-30a?+?vectors were digested by Nde I and Xho I double enzymes and linked to construct pET-30a?+?-TgPRF recombinant plasmid.The pET-30a?+?-TgPRF was transformed into E.coli XL10-GOLD competent cells.Positive colonies were screened and identified by PCR and sequencing.The correctly sequenced plasmids were transformed into E.coli BL21-?DE3?expression bacteria.The expressed products were analyzed by SDS-PAGE and Wester blotting after IPTG induction.The expressed products were purified by Ni-NTA affinity column and ion exchange chromatography.Polyclonal antibodies were prepared by immunizing New Zealand rabbits with protein.Co-culture system of mouse embryonic fibroblasts?BALB/C-3T3?and tachyzoites of T.gondii?RH-GFP?was established in vitro,and firstly observed the killing effect of TgPRF polyclonal antiserum?uncompleted?on normal cells and tachyzoites by fluorescence microscopy.Then,the effect of polyclonal anti-serum of TgPRF at different concentrations on the proliferation of T.gondii tachyzoites in BALB/C-3T3 in vitro was observe by fluorescence microscopy.The experiment was divided into four groups:Model group A supplemented with tachyzoites of T.gondii 8�10³;intervention group C:divided into C1^C4 group,TgPRF polyclonal antiserum was added at a final concentration of 1:40,1:80,1:160,1:320,respectively,each group challenged with tachyzoites of T.gondii 8�10³;negative control D:supplemented with tachyzoites of T.gondii 8�10³ and rabbits pre-immune serum?final concentration,1:40?.Three replicate?n=3?were set for each group and incubated in a incubator of 5%CO₂ at 37?for 96 h,and took photographs at12 h intervals for observation.The proliferation of the parasite was analyzed by image pro6.0 software,and the proliferation ratio and inhibition rate of the parasite were calculated.The effect of TgPRF antibody intervention on the growth and apoptosis of Toxoplasma gondii?RH?infected cells was further monitored by IncuCyte ZOOM?system.The model group A'and the intervention group C1',C2',and C3'were set up,and the normal control group?N?and 1:40 TgPRF antibody control group?B?was added.Three replicate?n=4?were set for each group mentioned above and the volume of each well was adjusted to 200?l.Results:1.The total RNA of T.gondii was successfully extracted,and three bands were shown by gel electrophoresis.The length of the PCR product was 510 bp.The recombinant plasmid was identified by specific primers and universal primers and sent to the company for sequencing.The results showed that pET-30a?+?-TgPRF was successfully constructed.SDS-PAGE analysis showed that the soluble target protein was mainly expressed in the supernatant of the ultrasound bacterial solution,which was soluble expression and the Mr was about 26 kDa,which was consistent with the expectation.2.Optimized expression conditions and found that the optimal IPTG induction concentration was 0.2 mM,the induction time was 10 h,and the induction temperature was 37?.A large amount of the recombinant protein was collected and purified by Ni-NTA affinity chromatography and anion column.It was found that the purity of TgPRF recombinant protein was more than99.5%after elution with 37.5 mM imidazole and 0.2 M sodium chloride.3.The purified recombinant protein was emulsified with Freund's adjuvant to immunize New Zealand rabbits to prepare polyclonal antibody.The titer of antibody was more than 10⁶ by ELISA test.Western blotting results showed that TgPRF antiserum could specifically recognize the corresponding antigen.4.The TgPRF antiserum without complement was non-toxic to cells and Toxoplasma gondii by in vitro experiments.In vitro intervention experiments showed no significant difference between group A and group D?P=0.382?,which was higher than that of group C?P<0.05?.The parasite proliferated slowly before 48 h,and then began to proliferate fast.The proliferation ratio of group A was 1.091�0.137 at 48 h,significantly higher than group C1^C3?P<0.05?,and there was no significant difference between group A and group C4?P=0.107?.At 60 h after infection,all groups continued to proliferate and the intervention group showed a higher inhibition on the proliferation of T.gondii.The inhibition rates of group C1^C4 were 78.13%,69.01%,64.75%and 50.92%,respectively.The tachyzoite proliferation ratio of group A was significantly higher than that of group C1^C4?P<0.05?at 72 h,and the inhibition rate of the intervention group was basically stable.At 84 h after infection,the tachyzoite proliferation ratio of group A increased to 20.539�1.414,which was significantly higher than that of the intervention groups?P<0.01?.The tachyzoite proliferation ratio of group C4 was 9.546�1.143,which was higher than that of other intervention groups C1³?P<0.05?.There was no significant difference between group C1^C3?P>0.05?.5.IncuCyte ZOOM^TM assay showed that there was no significant difference in the cell confluence between normal control group?N?and C1'?P=0.696?,which was significantly higher than that of C2',C3',while that of A'group was significantly lower than that of other groups?P<0.05?.At the end of the experiment,the proliferation of cells in group A was severely inhibited and the damaged seriously.The cells in group N covered the whole field of vision,while only a small part of cells in group C1'was missing.The C2'and C3'groups had obvious intercellular spaces with varying degrees of local missing.There was no significant difference in apoptosis between the intervention group and the infection control group?P>0.05?,in which the apoptosis of the infection control group was scattered throughout the field of vision,while the apoptosis of the intervention group was mostly concentrated in the tachyzoite proliferation region.Conclusion:1.Prokaryotic expression system of pET-30a?+?-Profilin recombinant protein was successfully constructed and TgPRF recombinant protein was expressed.2.High purity TgPRF protein was obtained by Ni-NTA affinity and anion chromatography.Rabbit anti-TgPRF antibody was successfully prepared.3.In vitro experiments showed that the TgPRF antibody of Toxoplasma gondii inhibited the proliferation of tachyzoites in BALB/C-3T3 with a dose-dependent manner,and the mechanism may be closely related to the induction of apoptosis of infected cells.