Study On The New Vaccine Of Malaria

Malaria is a major global health problem for which effective control measures are urgently needed.Considerable effort has been focused on the development of effective vaccines against the causative parasite and protective vaccine trials are now being reported.Due to the relative poverty and lack of infrastructure in malaria-endemic areas,a successful immunisation strategy will depend critically on cheap and scaleable methods of vaccine production,distribution and delivery.One promising technology is transgenic plants,both as a bioreactor for the vaccine-manufacturing process as well as a vector for oral immunisation.In this study, we investigated the feasibility of using transgenic plants to produce C-terminal region of the merozoite surface protein(MSP1-42) of P.falciparum(3D7) with antigenicity and immunogenicity,and some important results were described as following.1.The rasp1-42 gene of Plasmodiun falciparum was synthesized by two-step PCR method from 23 pairs of primers with eodon modification aecordding to rice codon preferences.2.To check if the synthetic msp1-42 gene could be expressed in E.coli and to obtain MSP1-42 protein for further investigation.The 12 amino codon following the initiation codon were modified according to the E.coli codon preferences to generate Emsp1-42,then the Emsp1-42 was inserted into pET32a(+) to construct expressing plasmid pET32amsp.Rosetta gami(DE3) transformed with pET32amsp could produce soluble eMSP1-42 protein when induced by IPTG at 25℃,SDS-PAGE and Western blot analysis showed that the eMSP1-42 could be purified without other proteins and could be recognized by conformational specific monoclonal antibody mAb5.9,rabbit sera immunized with PfCP2.9 and pooled sera of patients infected with P.falciparum.Anti-eMSP1-42 rabbit sera prepared could recognize the nature protein by IFA,and the inhibition of parasite growth in vitro on heterologous parasite (P.falciparum Hcc1/HN isolate) of the three rabbits sera at the concentration of 10% and 20%were(51.9±24.2)%,(29.4±8.6)%,(86.7±7.4)%and(93.3±7.5)%, (65.3±10.6)%,(96.4±1)%,respectively,indicating that the eMSP1-42 could be expressed in Rosetta gami as soluble recombinant protein with antigenicity and immunogenicity.3.To investigate the possibility of producing subunit antigen of Plasmodiun falciparum in rice seeds,the modified C-terminal region of the major merozoite surface protein(Rmsp1-42) gene of P.falciparum 3D7 strain was synthesized and then expressed in rice seeds under the control of the rice seed specific GluB-4 promoter.The introduction of the Rmsp1-42 gene into rice genome was performed by PCR.Recombinant protein analysis indicated that the fused Rmsp1-42 gene was specifically expressed in rice seeds with about 189.56μg/g dried grain(10.62 mg of rMSP1-42 protein purified from 56 g powder of dried grain).It is estimated that the accumulation of rMSP1-42 protein in the seeds is about 1.56%of total soluble protein. Western blot and ELISA analysis showed that the rMSP1-42 could be recognized by eonformational specific monoclonal antibody mAb5.2,rabbit sera immunized with PfCP2.9 and pooled patient sera from malaria endemic area.Anti-rMSP1-42 rabbit sera prepared could recognize the nature MSP1-42 protein by IFA,and the inhibition of parasite growth in vitro on P.falciparum Hcc1/HN isolate carrying heterologous MSP1-42 of the three rabbits sera at the concentration of 10%and 20%were (18.2±8.5)%,(47.7±7.5)%,(40.6±9.7)%and(77.3±7.5)%,(87.6±4.7)%,(87.2±3.7) %,respectively,indicating that the rMSP1-42 could be expressed in rice seeds with antigenicity and immunogenicity.This study demonstrates the potential for producing efficacious malarial vaccines in transgenie rice.4.Try to introduce the msp1-42 into chloroplast genome of tobacco for expression of recombinant protein MSP1-42.Forward and reverse primers,which had been adjusted to tobacco codon preferences were used for generating Cmsp1-42 gene from pBluntmsp,then a chloroplast expression vector LRrrmsp was constructed and bombarded into leaves of tobacco by a He particle delivery system.Media containing 500 mg/L spectinomycin were used to select spectinomycin resistant plant.PCR analysis proved that the Cmsp1-42 gene had successfully been inserted into the chloroplast genome.Multiple PCR analyses showed that the chloroplast genome were heterogenous after 3 turns of selection under 500 mg/L spectinomycin.ELISA failed to detect the cMSP1-42 protein from the leave total soluble protein.It requires further analysis to see whether the cause of the failure is the un-stability of mRNAs or the degradation of cMSP1-42 protein.