Purification Process Of The New Chimeric Antigen (M312) Of Malaria And Its Immunogenicity Enhancement Study

Malaria is an ancient and infectious disease,is one of the most serious in human populations.This study aimed at solving the problems in downstream processing of a new multi-epitope chimeric antigen of Plasmodium falciparum,M.RCAg-1(M312 in short),which could be used for development of a malaria vaccine.Research efforts included solving the problem of instability in the separation and purification of M312,establishment of large-scale purification of M312.Also a bioconjugate vaccine based on M312 was prepared to increase the immunogenicity,which provides an engineering foundation for the development of malaria vaccine.Major novelties are as follows:(1)The reasons for the instability of M312 in separation and purification were discovered and the countermeasures were established.By the addition of imidazole and EDTA as chaperone of separation and purification,the degradation of the target protein by the presence of protease(s)in the initial extraction solution after cultivation and disintegration of the E.coli was successfully depressed,and the yield of separation and purification were improved.However,the effect of imidazole as the chaperone has not been reported yet.Its possible mechanism for protection of the target product is the coordinate chelating with metalloprotease(s)through the imidazole ring.Combination with EDTA,imidazole could denature the protease(s)and protect M312 from being degraded.(2)A complete process was designed and established for separation and purification of M312,featuring combination of metal-chelate chromatography and high-performance gel filtration chromatography as major steps.The process was scale-up to produce gram quantity of M312 in sterile clean workshop.The end product reached 95%purity,with 600 mg/L(culture medium)total recovery.The residuals of endotoxin content,host cell proteins,host cell DNA met with the standard of Food and Drug Administration of the US.The structure of M312 was characterized.Mass spectrometry revealed the molecular weight of M312 was consistent with the theoretical value.Circular dichroism indicated that the secondary structure consists of mainly disordered random coil.Immunization of mice with the M312 and Freund's adjuvant could generate large quantity of antigen-specific antibodies.Furthermore,the M312 prepared was stable at 4? for up to 6 months.(3)A strategy for enhancement of M312's immunogenicity was attempted.There exist free cysteine(s),so a conjugate antigen was designated to link a free cystine residual of M312 with an amino group of tFL,by the crosslinker of SC-PEG-MAL to carry out the coupling of the two proteins.The reaction was carried out under mild conditions,and the conjugate,tFL-PEG-MAL,was separated from the residual crosslinker by ion exchange chromatography.And then the final product M312-PEG-tFL was purified by high-performance gel filtration chromatography.SDS-PAGE showed a purity of about 90%of the conjugate product with 50%mono-M312-PEG-tFL conjugate.Circular dichroism revealed the conjugate retaining the secondary structure of M312 and tFL.After boosting immunization in mic,the M312-PEG-tFL elicited much higher anti-M312 antibody titer and stronger humoral immunity than M312 alone.Compared with M312,the M312-PEG-tFL conjugates enhanced the proliferation index,lymphocyte activation and memory T cell generation.IgG subclasses of sera and cytokines analysis of splenocytes showed that conjugation with tFL could trigger the Thl polarization,while the antigen alone predominantly induced a Th2-biased immune response.Furthermore,a more efficient innate immune response was provoked by the M312-P5k-tFL,as determined by the detection of antigen-specific TNF-a secretion by splenocytes indicating that the M312-P5k-tFLcould elicit more efficient innate immune responses while the tFL in the conjugate retained the function as an agonist of TLR5.