Sequence Analysis Of Toxoplasma Gondii GRA4?GRA7?GRA20?ROP20 And Immunopotency Of Recombinant Lactobacillus Plantarum Expressing GRA4 And GRA7

Toxoplasma gondii is an opportunistic parasite for all warm-blooded animals,which can cause the toxoplasmosis by living in cells.T.gondii infects the host by the contaminative food and water mainly,also has the vertical transmission.For the pregnant women,the infection will lead to abortion or fetal malformation or stillbirth.According to the statistics,1/3 of the world's population has infected with T.gondii,and the harm for livestock industry is also very significant,so in medicine and veterinary has a widely attention,but no efficient drugs can eliminate T.gondii,so the studies for efficient vaccine has been the hot topic in recent years.The lactic acid bacteria are found widely in nature,which is one of the important members of probiotics.It can inhibit the growth of harmful bacteria in humans and animals,regulate the balance of bacteria and the stability of pH in the intestinal,which maintains the homeostasis of the body.Lactic acid bacteria,as a kind of non-pathogenic bacteria,has the adjuvant activity,which can not produce endotoxin.The bacteria which expresses the exogenous protein can be directly applied via oral administration.As an ideal transformation and expression system,it has a broad application.The present study examined the sequence variation in the dense granular proteins4,7,20(GRA4,GRA7,GRA20)and rhoptry protein 20(ROP20)genes of T.gondii.First,PCR was used to amplify these genes of T.gondii from different hosts and geographic origins.Then the amplicons were cloned and sequenced.The sequences were aligned with Clustal x 1.83.Finally,the phylogenetic tree was constructed using the maximum likelihood method(ML),neighbor joining(NJ)and maximum parsimony(MP).The result was as follows.Among 12 T.gondii isolates collected from different hosts and geographical regions,the GRA4 and GRA7 genes were966 bp and 712 bp in length,and the sequence variations in nucleotides were 0~2.4%and 0~2.7%,respectively.The complete GRA20 gene sequence was 1586 bp in length among 16 T.gondii isolates from different hosts and geographical regions,the sequence variations was 0~7.9%,however,removing the type III strains(CTG,VEG),the sequence variations became very low,only 0~0.7%.Full length ROP20 genes were 1659 bp and had a sequence variation of 0~1.4% in 18 T.gondii isolates from different hosts and geographical regions.The phylogenetic tree indicated that the GRA4 gene can't reflect the genetic relationships between the 12 T.gondii isolates,however,the GRA7 gene can make 3 major clonal lineage types(type I,?and type III)into their respective clusters,the GRA20 gene can differentiate 2 major clonal lineage types(type I and type III)into their respective clusters,and the ROP20 gene can differentiate between the clonal lineage genotypes I and ToxoDB #9.The study also evaluated the immune effect of recombinant Lactobacillus plantarum expressing GRA4 and GRA7 gene of T.gondii.First,according to the GRA4?GRA7 gene sequences of the GT1 strain which were available in ToxoDB,the primers with the restricted enzymes Nco I and Xba I were designed by the Primers 5.0,and the His tag was introduced into the C end of the two proteins.Then the genes were amplifyed by PCR,cloned and sequenced.The fragments of GRA4 and GRA7 were transformed into the shuttle expression vector pSIP-409 of the Escherichia coli and Lactobacillus plantarum.After that,the recombinant expression vector pSIP-409-GRA4 and pSIP-409-GRA7 were constructed.The recombinant plasmids pSIP-409-GRA4 and pSIP-409-GRA7 were transformed into Lb.plantarum NC8 by electroporation,then after the identification by double enzyme digestion and PCR amplification,the recombinant plasmids were analyzed by Western-blot.After the recombinant Lb.plantarum expressing GRA4 ? GRA7 were generated successfully.Then the BALB/c mice were immunized with the recombinant Lb.plantarum by oral administration,while three control groups of the blank group,the blank milk powder group and the Nc8-pSIP-409 group were set up.Immunization program was that the mice were immunized three times weekly for two weeks,and after an interval of a week,the groups of mice were boosted twice.Every time per mice was immunized with the bacteria of 1.0×109cfu/200?L.Then some humoral and cellular immune parameters were tested.42 days after the first immunization,mice were infected with T.gondii RH(103 tachyzoites),and the time of death was recorded.Results showed that the mice which were immunized with the recombinant Lb.plantarum expressing GRA7 can produce higher level of Ig G,IgG2 a,IFN-? and CD3+CD4-CD8+,CD3+CD4+CD8-T lymphocytes,which represent the Th1 type immune response.However,the recombinant Lb.plantarum expressing GRA4 has a weak immunogenicity.The average survival time of control group is 6.1d,the mice immunized with the recombinant Lb.plantarum expressing GRA7 have prolonged the survival time to 6.8d,but no significantly different from the control groups,and immunization with the recombinant Lb.plantarum expressing GRA4 did not prolong the survival time of mice.This study was the first to use the recombinant Lb.plantarum expressing GRA4?GRA7 to immunize mice orally.The results indicated that the recombinant Lb.plantarum expressing GRA7 can induce partial protection against T.gondii,which provides a new idea for the future development of vaccines against T.gondii.