Expression And Immunology Study Of Subunit Vaccine FHbp In Escherichia Coli And Arabidopsis Thaliana

Meningitis is the world-wide infectious disease caused by Neisseria meningitidis, with the characteristics of high mortality and high infectiousness, especially for the six month to 2 year old baby and the permanent neurological sequelae will be left even cured.At present, the most effective way for preventing Meningitis is still vaccination. There are mainly five types of serotype and capsular polysaccharide conjugate vaccines for four of them including A, C, W and Y have been developed and approved for the application except for serotype B. However, as the polysaccharide structure of serotype B(MenB) is homologous with sialic acid of human tissue and unable to cause an immune response. Therefore, the effective polysaccharide vaccine is still lacking. The appearance of fHbp as the main candidate antigen provides an ideal channel for the development and application of meningitis B(MenB) vaccine.In this study, the fHbp gene isolated from pathogenic strains The first affiliated hospital of wenzhou medical university of Asian MenB and then prokaryotic expression vector, pET-22b-fHbp, was constructed after bioinformatics analysis of the gene sequence. The vector was transferred into BL21(DE3) cells of E. coli using heat-shock method and engineered stain was finally gained. It is confirmed that the maximum expression level of fHbp protein was achieved when induced at the condition as followed: inducing temperature is 37°C, concentration of ITPG is 500 mM and induced for 8h. Moreover, the fHbp protein is not only soluble but accumulated up to approximately 20% of the total soluble protein. The purified product from E. coli was obtained by means of Affinity chromatography with Ni2+ Sepharose Fast Flow and named as E-fHbp. The optimization of fHbp sequence was carried out according to the codon bias of Arabidopsis thaliana and then the optimized rfHbp gene was synthesized artificially. A plant expression vector with optimized rfHbp gene, namely pPhaP3301-rfHbp, was constructed, in which the rfHbp gene was driven by the promoter of PhaP of Phaseolus vulgaris. The vector was transferred into Arabidopsis via Flora-dip method and the less then 300 resistant plants were obtained after spraying with 1% glufosinate and further confirmed to 288 of them be positive by Aurum labeled immunoassay(PAT/bar strip tests). PCR analysis was employed to test the existence of rfHbp genes in plants, and a total of 87 plants were proved to be transgenic. The target protein isolated from pods of those 87 T1 plants were tested by ELISA to determine the expression of fHbp and five out of them were verified to accumulate more than 0.1% of pod TSP. Consequently, three plants, rHF-10, rHF-22 and rHF-54, were confirmed to integrate one copy of fHbp gene into the genome by Southern analysis. The highest level of recombinant protein, 0.349±0.072% TSP, appeared in the seeds of rHF-22 plant, which was proved by Western and ELISA. Purified recombinant fHbp protein from transgenic plants, named as A-fHbp, was achieved by affinity chromatography with Ni2+ Sepharose Fast Flow.The above two purified recombinant fHbp proteins were used for the immune experiments. The immunogenicity difference was compared between two vaccine candidates, E-fHbp derived from E. coli and A-fHbp derived from A. thaliana, via intraperitoneal injection. The results showed that the serum specific antibody titers induced by E-fHbp and A-fHbp were 2×104 and 4.8×104, respectively, and enable to induce SBA activity in inphan rabbit. Oral immunization experiment of two antigens were conducted in BalB/c mice, and the results showed that neither of them could induce the mucosal IgA in the immune system. Toxic test to mice by Neisseria meningitidis showed the protection rates are 76.67% and 80% for E-fHbp and A-fHbp respectively, and there was no significant difference between the two proteins in the protective effect as the vaccine candidates.This is the first report that the fHbp protein was expressed and purified in the seeds of transgenic plants, and the comparison of the two different sources of fHbp proteins in immunology as well. The results showed that there was no significant difference between the two kinds of proteins, and both of them has potential as vaccine candidates. These results provide supports for production of protein subunit vaccine via plant bioreactor, especially offered the important theoretical basis for the development and application of vaccine candidates for B subtype of Neisseria meningitidis.