| Lyme disease,which is caused by Borrelia burgdorferi,is the most common tick-borne disease in the world.The main vector of Lyme disease is ticks,which mainly grow on the top of plants 20 to 30cm from the ground.People without protective measures went thro μ gh the grass with the growth of ticks may lead to lyme disease for a tick bite,so the disease is relatively common in forest and grassland areas in Northeast,Northwest and South China[1-4]The symptoms of lyme disease in humans are mainly divided into early,middle and late stages,and the early stage is characterized by typical chronic migratory erythema(EM).Other early symptoms may include fever,headache and fatigue.If left untreated,patients can lead to the middle or late symptoms:mainly including neurological damage,joint pain,accompanied by serious headache,neck stiffness[5],prolonged illness may lead to post-lyme disease syndrome(PLDS)[6],which characterized by mild to severe musculoskeletal pain,fatigue,or difficulty in concentration and memory.The study of an effective vaccine is an important measure to control lyme disease.But until now no lyme disease vaccine has been successfully developped for humans.The vaccine of lyme disease mainly includes subunit vaceine,DNA vaccine and so on.Considering the polymorphism and heterogeneity of the gene structure and antigenicity of lyme disease spirochetes,there are obvious differences between strains of different genotypes and it is difficult to obtain good cross mmunoprotection,the solution for this problem is to develop polyvalent subunit vaccines for the main pathogenic genotypes or subtypes in different regionsThe study aims to clone and express the OspA peptide(126-274aa)of Chinese Borrelia garinii strain PD91(PD91-rOspA-pep),and explore the immune protectivity of recombinant OspA peptide combined with recombinant OspC proteins of B.garinii and B.afzelii genotypes of Lyme disease in China.,So that we can provide basic data for the development of subunit vaccine for lyme disease in ChinaThe first section:The 126-274aa OspA peptide gene of Chinese Borrelia garinii strain PD91 was amplified by polymerase chain reaction(PCR)and cloned into prokaryotic expression vector pET-30a to construct the recombinant plasmid pET-30a-OspA-pep,And then the recombinant plasid was transferred into competent Escherichia coli cells BL21(DE3),induced expression by IPTG,the protein was purified with Ni-IDA resin chromatography,and analyzed by SDS-PAGE electrophoresis and Western blot assay;New Zealand rabbits were immunized with different doses of rOspA-pep(20μg,30μg,40μg,50μg,60μg,80μg,100μg).The titers of specific antibody IgG in serum of rabbits before and after immunization were detected by indirect immunofluorescence method(IFA).The optimal immune dose was determined according to the antibody titer after innunization.In vitro neutralization test was used to detect the immune protection of rOspA-pep protein.The optimal dose of rOspA-pep was used to immunize rabbits to observe the changes of the antibody titerThe second section:New Zealand rabbits were immunized with OspC combination protein of PD91 and FP1 strains.The group contained 7 dose groups of 20,30,40,50,60,80 and 100 μg,and 1 blank control group(0 μg),with a total of 8 dose groups.The control group was injected with PBS.Indirect immunofluorescence method(IFA)was used to detect titers of specific antibody(IgG)in rabbit serum before immunization and after immunization for 15,30,45 and 60 days respectively.The optimal immune doses of rOspC were determined according to the antibody titer after immunization.In vitro neutralization test was used to detect the In vitro antibacterial ability rOspC protein.Ten New Zealand rabbits were selected to immunize rOspA-pep+rOspC proteins at the optimal dose.They were immunized for the first time on the first day and strengthened on the 30th day.The IgG antibody in the serum of the 15th,30th,60th,90th,120th,150th and 180th days after the first immunization was detected by IFA method.The antibody titer curve was drawn to observe the changes and duration of antibody and determine the best time to attack bacteria.In addition,Sixteen New Zealand rabbits were divided into two attack dose groups,each group has 5 rabbits which were randomly selected as the attack group and immunized with the optimal dose of rOspA-pep+rOspC protein,and the other 3 rabbits were used as blank control group and subcutaneously injected with PBS with the same volume as that of the attack group.After 60 days of the first immunization,the PD91 strains with 2×105 and 2×106/ml were subcutaneously injected into the bodies of the two groups of rabbits.The kidneys and bladders of rabbits were dissected for culturing and nucleic acid testing.DNA from these tissues was extracted on 5th week.Then the 250bp target fragment of the 5s-23S rRNA interval region of Borrelia burgdorferi was amplified by nested PCR to determine the positive sample.The data of experiments were collected and analyzed.The results of first section:The recombinant plasmid PET-30a-ospA-pep was successfully constructed and highly expressed in host bacteria(E.coli B21).Western blot showed that rOspA-pep had obvious antigen-antibody reaction with polyclonal antibody against B.garinii PD91 strain in the first section.IFA results showed the titer of IgG antibody in serum of rabbits immunized with rOspA-pep protein increased significantly.(up to 1:2480),40μg was the optimal dose.The neutralization rate of antibodies of 40 μ g rOspA-pep against 106/ml B.garinii and B.afzelii representative strains PD91 and FP1 was 100%,for 107/ml FP1 was 100%,and for 107/ml PD91 was 60%.After immunization with 40 μg rOspA-pep twice on the first day and the 30th day,the titer of specific antibody IgG in rabbit serum reached a culmination within 45-60 days,and the duration of the antibody was about 3-4 months,then the antibody titer began to descend.The results of second section:After immunizing New Zealand rabbits with a rOspC combination of PD91 and FP1,the IFA results showed that the titer of serum IgG antibody immunized with rOspC was significantly increased.30pg was the optimal immune dose of rOspC.The neutralization rate of antibodies of 30 μ g rOspC against 106/ml B.garinii and B.afzelii representative strains PD91 and FP1 was 100%.After immunization with 40μg rOspA-pep twice on the first day and the 30th day,the titer of specific antibody IgG in rabbit serum reached a peak within 45-60 days,and the duration of the antibody was about 2-3 months,then the antibody titer began to decline.In vivo infection test,the results showed the nucleic acid detection of the immune group and the control group was negative when they were attacked by 2 × 105 CFU living bacterium,and there was no difference between the immune group and the control group.It was suggested that the antibody produced by the rabbit itself can neutralize the pathogen and play a protective role when it was attacked by a low dose of Lyme disease spirochete;In the challenge dose of 2×106cfu group,all the nucleic acid tests in the immune group were negative,while tissues of 1 rabbit in the control group had been tested positive,which were confirmed by sequencing.It was suggested that the protein combination of 40 μg rOspA-pep and 30μg rOspC has certain immune protection to rabbits when the attacking bacteria was 2×106 CFU.The 126-274aa OspA peptide fragment of chinese B.garinii strain PD91 has good immunogenicity and in vitro neutralization ability,and can be used as a candidate component of the second generation subunit vaccine in China.This study firstly demonstrates that the combined components of 40 μg rOspA-pep(PD91)and 30μg rOspC(PD91+FP1)can protect New Zealand rabbits from infection when they are attacked by high dose(2 × 106 CFU)Borrelia burgdoferii strains,This study provides basic data for the design of chinese multivalent subunit vaccine based on OspA peptide and ospC for Lyme disease.. |
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