| The expectant mothers living in endemic areas suffer from high risk of malaria.In sub-Saharan Africa ,there are twenty-five million of them who are exposed to this disease.The clinical consequences are abortion, maternal anemia and LBW. PAM can result in fetal morbidity,and cause 75,000-200,000 deaths of infant every year in the world. The accumulation of PRBC in the placenta caused PAM.This is mediated by the interaction between CSA in the placenta and a unique PfEMP1 expressed by parasite on the surface of the PRBC.This distinct PfEMP1 named VAR2CSA is encoded by var2csa which belonged to var gene family and is a large protein (350 kDa) consisting of six DBL domains.The level of anti-VAR2CSA IgG is associate with the protection against PAM.It indicated that VAR2CSA is a leading PAM vaccine candidate that can protect women in endmic areas.However, the challenge for VAR2CSA based vaccine is that several DBL domains have the CSA-binding activity. And it is very difficult to express and purify the VAR2CSA due to its huge size. Thus, it is very necessary to define which domain plays the major role in CSA-binding and immune responsing.In this study,we cloned,expressed and annalyzed the difference of CSA- binding activity as well as immunologic recognition among different domains.Six specific primer pairs were designed and synthesized according to the sequences. And six DBL domains were amplified by PCR and cloned into the vector pMD18-T. The recombinant plasmids were identified by PCR ,enzyme digestion and sequencing. The inserts with correct sequences were subcloned into the prokaryotic expression vector pET-22b, then transformed into E.coli BL21 (DE3) and induced by IPTG. The recombinant proteins were purified by His GraciTrap and further identified by SDS-PAGE and Western blot. The relative molecular mass of all the domains were as expected respectively. The purified proteins had been confirmed with immunogenicity by Western blot.Activity of CSA-binding of different recombinant DBL domains was assayed by ELISA method. Plates were pre-incubated with CSA (overnight at 4℃). After washing three times by washing buffer, the wells were blocked with 3% BSA for 1h at 37℃. Then, A dilution series of each protein were added and incubated for 1h at 37℃. After washing three times by washing buffer, His-tag(2A8)mouse mAb diluted 1:5,000 in Tween buffer was added to each well and incubated for 1h at 37℃. After washing three times by washing buffer, Anti-Mouse IgG diluted 1:20,000 in Tween buffer was added to each well and incubated for 1h at 37℃.The assay was finished with three times washing and developed using pNPP substrate for 30 min. Absorbance was measured at 405nm.The results of adhesion experiment indicated that OD405 values of DBL5 domain with different concentration were significantly higher than that of DBL4 and DBL6 and were 2.1 times than that of Sj23. The OD405 values of all DBL domains are higer than that of Sj23. It suggested that DBL5 domain has high binding affinity with CSA, though all the DBL domains have the CSA-binding activity.Experiment of immunologic recognition of different recombinant DBL domains were measured by ELISA method in panels of individual plasma samples from PAM patients. This assay was performed basicly as the above but pre-incubating with different recombinant DBL proteins and adding patients serum, Anti-Human IgG instead of recombinant protein and Anti-Mouse IgG and without adding His-tag(2A8)mouse mAb. The results of immunologic recognition experiment were analyzed by using SPSS17.0.The results indicated that levels of IgG specific for recombinant proteins corresponding to the DBL4,DBL5,DBL6 and Sj23 were significantly different.Median of DBL5 was significantly higher than others.It suggested DBL5 may play the major role in immune response.The results obviously have direct implications for VAR2CSA-based vaccine development but also for the development of vaccines based on other members of the PfEMP1 family.
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