| Babesia belongs to the Apicomplexa SporozoaPiroplasmida and is an important tick-borne protozoan parasite in human or other mammalian erythrocytes that causes zoonotics babesiosis.Plasmodium and Babesia both belong to the same family of Apicomplexa,parasitize host erythrocytes,have similar subcellular structure and secrete conserved proteins associated with invasion,which play an important role in invading host cells.Studies in the literature have shown that pre-infection with Babesia can inhibit Plasmodium infection.Based on this,we constructed a mouse model of co-infection between Babesia microti(B.microti)and Plasmodium berghei(P.berghei)to evaluate the effect on Plasmodium berghei co-infection in mice.Later,according to the high-throughput screening of the protein microarray,we obtained Babesia microti erythrocyte membrane-associated conserved protein(renamed as Bm8 in this study),after cloning and expressing the protein,the antigenicity of the protein and the localization of the parasite were analyzed and the peptide fragments were synthesized.The aim of this study was to evaluate the immunoprotection of the polypeptide in the infection model of Babesia microti and Plasmodium berghei mice,and to provide reference for the vaccine targets of Babesia microti and Plasmodium berghei.Part 1 Construction of a co-infection model between Babesia microti and Plasmodium bergheiObjective:To investigate the effects of different infection times of Babesia microti on parasite loads in mice reinfected with Plasmodium berghei.Methods:Two different infection models were established.1)Simultaneous infection model.The mice in the experimental group were simultaneously infected with Babesia microti and Plasmodium berghei.The mice in the control group were only infected with the same amount of Plasmodium berghei and normal mouse blood.The time of the first infection with Plasmodium berghei was calculated as Day0.On the 3rd,6th and 14th days after infection,the infection of Plasmodium berghei in mice was detected by blood smear staining and real-time quantitative PCR.2)Babesia microti pre-infection model.The mice in the experimental group were pre-infected with Babesia microti for 21 days.The same amount of normal mouse blood was injected intraperitoneally.After 21 days,both groups of mice were infected with Plasmodium berghei,and each mouse was intraperitoneally injected with about 1×107 iRBCs(infected mouse blood dilution about 100 μL).The infection time of Plasmodium berghei was Day 0,and the Plasmodium berghei infection in mice was detected by blood smear staining and counting as well as real-time quantitative PCR on the 3rd,6th and 9th days after Plasmodium berghei infection.Results:1)Co-infection model.The concentrations of parasites in the co-infection experimental group(n=5)under the microscope on the 3rd,6th and 14th days of infection were(3.80±0.74)%,(46.75±2.16)%and(60.00±8.63)%,and the concentrations of parasites of the single infection control group(n=5)were(5.60±2.15)%,(51.00±8.64)%and(66.60±2.35)%.Compared with the single infection control group,the co-infection group showed a decreasing trend,but no statistical difference was observed.qRT-PCR data showed that on the 3rd,6th and 14th day of coinfection,the number of copies of Plasmodium berghei gene in co-infection group was lower than that in single infection control group,but no statistical differences were observed.2)Babesia microti pre-infection model.The microscopic concentrations of parasites in co-infection experimental group(n=5)were(3.38±1.06)%,(22.60±4.36)%and(56.00%±6.57)%on the 3rd,6th and 9th days of infection.The concentrations of parasites of the single infection control group(n=5)were(6.60±1.01)%,(28.80±4.30)%and(55.40±7.11)%.The concentrations.of parasites in the peripheral blood of the mice in the infection experimental group was lower than those in the single infection control group,and there was a statistically significant difference on the third day(**P<0.01).qRT-PCR data showed that the copy number of Plasmodium berghei in the mice in the co-infection experimental group was lower than that in the single-infection control group on the 3rd day of infection,and on the 3rd day(**P<0.01)there was a statistically significant difference.Conclusion:Both co-infection modes confirmed that the parasite load of P.berghei in the co-infection group was lower than that in the single-infection control group.The results indicate that Babesia microti co-infection can partially induce the immune protection of mice against P.berghei infection,suggesting that the immunity induced by Babesia microti infection may have a certain cross-immune protection against P.b erghei infection.Part 2 Cloning,expression and antigenic analysis of Babesia microti erythrocyte membrane-associated conserved proteinObjective:Cloning,expression,antigenicity analysis and cellular localization of Babesia microti erythrocyte membrane-associated conserved protein.Methods:1)High-throughput screening of the target protein Babesia microti erythrocyte membrane-related conserved protein Bm8 was performed using the previous protein chip,and the full-length gene fragment of Bm8 was amplified by PCR technology,and then the recombinant protein Bm8(rBm8)was obtained by cloning,expression and purification.2)IFA observed the localization of Bm8 in Babesia microti and Plasmodium berghei.3)ELISA evaluated the antigenicity of rBm8 to the sera of mice infected with Babesia microti and Plasmodium berghei.Results:1)The recombinant plasmid pGEX-6p-2-Bm8 was successfully cloned and constructed,and the recombinant protein Bm8 was obtained after prokaryotic expression and purification.2)ELISA showed that rBm8 specifically bound to the serum of mice infected with Babesia and and showed a strong immune response.3)IFA showed that Bm8 was mainly located in the merozoite membrane of Babesia microti and Plasmodium berghei.Conclusion:rBm8 with a molecular weight of 57 kDa was obtained by cloning,expression and purification.ELISA results confirmed that rBm8 could be recognized by mouse serum infected with Babesia microti and Plasmodium berghei.IFA showed that the protein was localized in the merozoite membrane of two protozoa Babesia microti and Plasmodium berghei,suggesting that the protein was a merozoite stage surface protein of two protozoa.Part 3 Induction of Babesia microti erythrocyte membrane-associated conserved polypeptides immune protection researchObjective:To explore the evaluation of the immune protection of Bm8 polypeptide active immunization and Bm8 polypeptide antiserum passive immunization in two mouse models infected with Babesia microti and Plasmodium berghei.Methods:1)Bm8 polypeptide containing 35 amino acids was synthesized after Bm8 was predicted and analyzed by antigenic linear epitope.2)Active immunization model was established,and BALB/c mice were actively immunized for the first time.The experimental group was immunized with Bm8 polypeptide(20 μg)and an equal amount of Freund’s complete adjuvant,and 40 μg Bm8 was used for the second and third time.The peptides were immunized with the same amount of incomplete Freund’s adjuvant.A total of three immunizations were completed.One week after the last immunization,ELISA was used to detect the Bm8 polypeptide antibody titer in the serum to evaluate the effect of active immunization.The control group was immunized with adjuvant only.After the experimental group achieved the expected immune effect,the mice were infected with Babesia microti or Plasmodium berghei,and each mouse was intraperitoneally injected with about 1×107 iRBCs(about 100 μL of infected mouse blood dilution).The first infection time was Day 0,in the model of Babesia microti infection,On the 3rd,6th,9th and 12th days after infection,the infection of Babesia microti or Plasmodium berghei in mice was detected by blood smear staining and real-time quantitative PCR.Hemoglobin concentration,weight,temperature changes and survival time of two groups of mice were observed at the same time.In the Plasmodium berghei infection model,the same method was used 3,6 and 9 days after infection to detect the Plasmodium berghei infection in mice and observe the survival time of mice.3)Passive immune model was established by intraperitoneal injection of Bm8 polypeptide antiserum(200 μL)to the experimental group BALB/c mice and normal rabbit serum(200 μL)to the control group mice,and the mice were infected with Babesia microti or Plasmodium berghei 24 hours later.Each mouse was intraperitoneally injected with about 1×107 iRBCs(about 100 μL of infected mouse blood dilution).On the 4th day of infection,two groups of mice were given a booster immunization(the same amount as the primary immunization).The first infection time was Day0,in the model of Babesia microti infection,On the 3rd,6th,9th and 12th days after infection,the infection of Babesia microti or Plasmodium berghei in mice was detected by blood smear staining and real-time quantitative PCR.Hemoglobin concentration,weight,temperature changes and survival time of two groups of mice were observed at the same time.In the Plasmodium berghei infection model,the same method was used 3,6 and 9 days after infection to detect the Plasmodium berghei infection in mice and observe the survival time of mice.Results:1)After the active immunization model,mice were infected with Babesia microti.qRT-PCR data showed that on the 9th days of infection,the Bm8 polypeptide immunized mice had lower Babesia microti gene copies number than those in the adjuvant group,the difference was statistically significant(**P<0.01)..The concentrations of parasites under the microscope in the Bm8 polypeptide immunization group(n=5)were(5.10±0.66)%,(43.60±6.28)%,(22.20±3.86)%and(1.40±0.58)%,respectively.The concentration of parasites in the adjuvant control group(n=5)were(5.80±0.92)%,(51.80±6.11)%,(29.40±4.58)%and(2.20±1.16)%.The data showed that the concentrations of parasites in the Bm8 polypeptide active immunization group was lower than those in the adjuvant group on the 9th day,the difference was statistically significant(*P<0.05).There were no significant differences in weight,temperature and hemoglobin concentration between the two groups.2)After the passive immunization model,mice were infected with Babesia microti.The qRT-PCR data showed that on the 3rd day of infection,the Bm8 polypeptide antiserum group had more Babesia microti gene copies than the normal serogroup.The difference was statistically significant(**P<0.01).The concentrations of parasites under the microscope in the Bm8 polypeptide antiserum group(n=5)were(4.10±2.05)%,(48.20±3.05)%,(9.80±0.74)%and(2.40±1.01)%,respectively.The concentrations of parasites in normal serum control group(n=5)were(5.30±0.92)%,(49.00±7.26)%,(10.40±3.00)%and(1.80±0.81)%.The data showed that there was no statistical difference in the peripheral blood concentration of parasites between the two groups of mice.Compared with the Bm8 polypeptide antiserum group,the hemoglobin concentration of mice in the normal serum group was lower on the 3rd and 6th day after infection with Babesia microti,and the differences were statistically significant(*P<0.05,**P<0.01).There was no statistical difference in the weight and temperature of the mice in the groups.3)After the active immunization model,mice were infected with Plasmodium berghei.qRT-PCR data showed that on the 9th day of infection,the number of Plasmodium berghei gene copies in the Bm8 polypeptide immunized group was significantly higher than that in the adjuvant group.The difference was statistically significant(**P<0.01).The concentrations of parasites under the microscope in the Bm8 polypeptide immunization group(n=5)were(2.50±1.41)%,(42.20±4.62)%and(62.80±5.11)%.The concentrations of parasites in the adjuvant control group(n=5)were(2.76±0.69)%,(44.20±2.63)%and(69.00±5.83)%.The data showed that there was no statistical difference in the peripheral blood concentration of parasites between the two groups of mice.There was no statistical difference in weight,hemoglobin concentration and survival curve data between the two groups of mice.On the 9th day of infection,the temperature of the mice in the Bm8 polypeptide immunized group was higher than that in the adjuvant control group(*P<0.05).4)Mice were infected with P.berghei after passive immunization model.qRT-PCR data showed that P.berghei gene copies of mice in the Bm8 polypeptide antiserum group reduced significantly on the 9th day of infection(*P<0.05)compared with those in the normal serum control group.The concentrations of parasites under the microscope in the Bm8 polypeptide antiserum group(n=5)were(1.72±0.57)%,(37.80±6.61)%and(56.60±5.31)%.The concentrations of parasites in the normal serum control group(n=5)were(2.14±1.06)%,(44.20±2.63)%and(64.20±2.85)%.The data showed that the concentrations of parasites in the peripheral blood of mice in the Bm8 polypeptide antiserum group were lower than those in the normal serum control group on the 9th day(*P<0.05).There were no significant differences in weight,hemoglobin concentration and survival curve data between the two groups.On the 9th day of infection,the temperature of the mice in the Bm8 polypeptide antiserum group was significantly higher than that in the normal serum control group(***P<0.001).Conclusion:The protective effect of Bm8 polypeptide in Babesia microti and Plasmodium berghei infection models was evaluated by constructing active immunization and passive immunization.The results showed that in the Babesia microti infection mouse model,active immunization with Bm8 polypeptide could inhibit the invasion of erythrocytes by Babesia microti on the 9th day of infection,resulting in a certain immune protection for infected mice.Passive immunization with Bm8 polypeptide antiserum inhibited the invasion of erythrocytes by Babesia microti on the 3rd of infection.In the mouse model of Plasmodium berghei infection,both active immunization with Bm8 polypeptide and passive immunization with Bm8 polypeptide antiserum inhibited the invasion of erythrocytes by Plasmodium berghei on the 9th day of infection,and produced certain immune protection in infected mice.In conclusion,Babesia microti erythrocyte membrane-associated conserved polypeptide can induce certain immune protection,suggesting that the Babesia conserved protein has the potential to become the target of Babesia or malaria vaccines. |
