Pilot Study On The Vaccine Development By T Cell Epitope Of Group Ⅰand ⅡAllergens From Dermatophagoides Pteronyssinus

Objective To construct the T cell epitope vaccine from group I and II allergens from Dermatophagoides pteronvssinus(Der p1, Der p2) via prokaryotic expression of the protein with plasmid pET-28a(+), and examine the effectiveness of the expressed protein in immunotherapy.Methods1) Recombinant DNA technology was used to insert the synthetic T-epitope antigen gene of Der p1and Der p2into pET-28a(+) plasmid for initial construction of pET-28a(+)-T1-8, between the two epitopes with "GPGPG" sequence interval, and recombinant vector was verified by sequencing and digested with restriction enzyme.2) To transform the recombinant expression vector pET-28a(+)-T1-8into E.coil BL21(DE3), and screen the positive clones by colony PCR.3) Followed by expression induction in E.coli BL21, SDS-PAGE and Western blot were performed to verify analysis the protein T1-8.4) Forty mice were randomly divided into four groups, namely negative control group(PBS group), positive control group(Asthma group), allergens rDer p1and rDer p2were used for specific immunotherapy(rDer p1/rDer p2group) and protein T1-8for specific immunotherapy(T1-8group). The extract of house dust mites(HDM) was used to establish the asthmatic models in BALB/c mice, PBS group always used PBS buffer.30minutes before spray inhalation in25d-27d, the mice of the two SIT groups were respectively injected subsutaneously with their therapeutic proteins for SIT, then collected the serum, the bronchoalveolar lavage fluid (BALF) and the supernatant of splenocyte cultures (SSCC). ELISA was used to assay the levels of IFN-y, IL-4, IL-10and IL-17in the BALF and SSCC, as well as serum levels of specific IgE, IgG1and IgG2a. The lung tissue sections were stained with haematoxylin and eosin(H&E) for pathological examination.Results1) Digestion by restriction enzymes BamH I/Not I showed that, the recombinant vectors pET-28a(+)-T1-8were successfully constructed.2) Colony PCR showed successfully transformed to E.coil BL21(DE3) state of feeling.3) The fusion protein was successfully expressed in E.coli BL21by SDS-PAGE and Western blot analysis.4) ELISA detection revealed, alleviated significant pulmonary inflammation in SIT groups with decreased IL-4, IL-17level in the BALF and SSCC and serum IgE, IgG1, yet increased IFN-y, IL-10and serum IgG2a levels as compared with Asthma group. The difference was significant(P<0.05). The lower level of IL-4, IL-17and IgE and higher IFN-y, IL-17and IgG2a in the T1-8group were obvious, compared to those in the rDer p1/rDer p2group (P<0.05).Conclusions The recombinant prokaryotic expression vectors pET-28a(+)-T1-8was constructed successfully and purified on a large scale. The recombinant protein T1-8, which were as vaccines for specific immunotherapy with asthma models, can alleviate effectively allergic inflammation of airway and lung in the mice, and it may be used as candidate vaccines for asthma.