| Malaria is one of three major infectious diseases to human health in the world; it is mainly popular in tropical and subtropical areas. According to the most recent data from WHO, there are approximately216million cases and0.655million deaths caused by malaria only in2010, an estimated3.3billion people were at risk of malaria in2010, and most of the deaths are children under five years of age and pregnant women in sub-Saharan Africa. Plasmodium falciparum is the most severe and deadly one among five species of malaria parasites that affect humans. Recently, though significant progresses have been obtained in Malaria Control, the emergence and spread of drug-resistant parasites and insecticide-resistant Anopheles mosquito vectors make traditional prevention and treatment of malaria a big hurdle, which enhance the demand of preventable and therapeutic malaria vaccine all over the world. Plasmodium have the characteristic of complex life cycles, antigenic stage-specificity, diversity, variation and the mechanism of avoid the host immune attack, and vaccines which based on single epitope haven’t made great progress in related researches. Therefore, development of a multiple antigens and epitopes vaccine against Plasmodium falciparum at different stages has become a major hotspot in the development of malaria vaccines.Previously, Our Lab has been exploring multiepitope vaccine against Plasmodium falciparum, we constructed a polyepitope gene library by using a novel approach named "’epitope shuffling" and selected a polyepitope chimeric protein M.RCAg-1with high immunogenicity and stability from the library, and finally we got the high expression genetic engineering bacteria. We also proved that M.RCAg-1had strong immunogenicity and its specific antibodies could inhibit Plasmodium falciparum growth in vitro when emulsified it with some adjuvants in both mice and rabbits. These results showed that M.RCAg-1might be used as a blood-stage candidate vaccine against Plasmodium falciparum with the value of further optimization and development. Nevertheless, after preliminary enlarge production, the quantity of recombinant protein was lower and M.RCAg-1sample could degrade or aggregate when stored it in-80℃within several weeks, which gave rise to difficulties in the following research. Therefore, get high purity and stability M.RCAg-1combinant ptotein sample is the key step in preclincal studies.On the basis of previous studies, we found out a purification process of M.RCAg-1by using five different combinations of Ni affinity chromatography, anion exchange chromatography and gel filtration chromatography to purify the target protein according to the physicochemical property of M.RCAg-1, and the optimal purification approach was selected in the light of final purity and yield of M.RCAg-1. Meanwhile, we selected Crystal Screen HT (HR2-130) Kit and mixed M.RCAg-1sample with the kit solution, and we had screened the most sutiable buffer for M.RCAg-1through the inverted microscope observation and12%SDS-PAGE electrophoretogram. Moreover, in view of disulfide bonds play an important role in aspect of protein correct folding, senior structure formation and maintain the biological activity, this paper also investigated the disulfide bonds determination of M.RCAg-1.This research mainly made the following progress:1. The expression means of M.RCAg-1in E.coli is soluble expression and its expression rate is greater than20%.2. We tried to find out the optimum purification process of M.RCAg-1and formed the foundation for the downstream large scale production and further investigation its immunity effect. In conclusion,206.68mg crude protein was obtained on the average of per litre LB liquid medium, target protein M.RCAg-1accounts for about20.92%of the crude protein, the final purification results showed that target protein is more than99%in purity and the recovery rate is greater than15%.3. We screened soluble and stable buffers for M.RCAg-1by using Crystal Screen HT (HR2-130) Kit. This study proved that no degradation was found more than six months when dissolved M.RCAg-1in PBS and stored it in-80℃.4. Disulfide bonds of M.RCAg-1were determined and analysed, the preliminary result is that M.RCAg-1molecule formed disulfide bond Cys215-Cys329and interchain disulfide bond Cys215-Cys215. |
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