Protiomic And Molecular Biology Analysis Of Silkworm (Bombyx Mori L.) Pupa Allergens

Silkworm(Bombyx mori Linaeus) is an important economic insect of China. However, silkworm have the capacility to induce allergic reaction in atopic individuals. In china, the pupa of silkworm is a kind of Chinese food and an important component of traditional Chinese medicine used for the treatment of hypertension and fatty liver. So pupa is also an important source of food allergen in China. Our study is focus on the study of the allergen of silkworm pupa.1. The SDS-PAGE and Western-blotting analysis of silkworm pupa allergen: The extract of silkworm pupa protein was carried on by high speed centrifugation. Then 12% SDS-PAGE was used to separate the protein content of the extract. 11 serum of silkworm pupa allergic patients were use to test it's immuno activity. In the result of SDS-PAGE, there are about 10 bands and 3 major bands show up around 25kDa. 5 bands (28, 35, 37, 65 and 90kDa) have the IgE binding ability with the serum of silkwom pupa allergic patients. The reaction rate of 4 of them (28, 35, 37 and 65kDa) reached 100%. The finding indicated that there are allergens exist in the extract and riched our knowledge of silkworm pupa allergen.2. Study the allergen of silkworm pupa by proteomic approaching: In this part, 2D, immunoblotting and MS approaching were used to inditify the allergen of silkworm pupa. The silkworm pupa protein extract was seperated by 2D gel. The 2D image pattern of silkworm pupa was built up. Then the protein in the 2D gel was transferred to membrane and incubated with the serum of silkworm allergic patients. The spots have the capacity to bind with IgE in the serum were analyzed by MS. The result indicated that totally 288 distinct protein spots were detected in the 2D gel. 13 of them have the capacity to binding with IgE in the serum of silkworm allergic patients. In the contrast, they do not bind with IgE in the serum of healthy people in another experiment. The result of MS analysis showed that they belong to 6 different protein classification including Vitellogenin, chitinase, 30K protein ,Triosephosphate isomerase, heat shock protein, chymotrypsin inhibitor.3. Cloning and prokaryotic expression vector construction of chitinase gene from silkworm pupa : In this study, our objective is to clone and prokaryotic expression vector const ruction of chitinase gene from silkworm pupa. The RT-PCR was applied to clone the full-length genes of chitinase gene from silkworm pupa and then the sequences were analyzed. The specific primers were designed. The ORF of chitinase gene of Bombyx mori Linaeus was subcloned into the expression vector p ET-28a. Chitinase gene from silkworm pupa was cloned and const ructed to prokaryotic expression vector. Chitinase allergen gene from silkworm pupa was cloned and constructed the prokaryotic expression vector successfully. It establishes the foundation for the recombinant allergen chitinase expression and the activity identification.4. The clone, expression, purification and immunoblotting of recombinant heat shock protein 20.8: The recombinant allergen could be use in clinical diagnosis and immunotherapy of allergic disease of individuals. In this part our study focus on exprssion the recombinant protein of heat shock protein 20.8. The gene of heat shock protein 20.8 was cloned and expressed in high yield in E. coli. The IgE binding activity was examined by western-blot. The sequence of heat shock protein 20.8 ampified by RT-PCR was mached with the sequence in database. The IgE antibodies from sera of patients showed positive reactivity to the purified recombinant heat shock protein. It's important to further basis experimental analysis of sikilworm pupa allergens and provide theory foundation for clinical diagnosis and treatment。Through this study identified the allergic protein of silkworm pupa, set up its 2-DE proteomics maps and allergen Western-Blotting map. Chitinase allergen gene from silkworm pupa was cloned and constructed the prokaryotic expression vector successfully. Then carried out the clone, expression, purification and immunoblotting of the recombinant heat shock protein 20.8.That has laid a solid foundation to allergens'proteomics and molecular biology basic research of silkworm pupa, as well as provided a theoretical basis to the specific diagnosis and treatment of silkworm pupa allergic diseases.