Differentia Expression Of Allergen In Silkworm(Bombyx Mori) Pupa

As a kind of high-quality proteogen,silkworm(Bombyx mori Lineaus)pupa has always been one of the favorite foods because of its rich nutriention,high-protein content and delicious taste.However,due to the existence of some allergens in silkworm chrysalis,a few people will suffer from allergies in real life,which not only restricts the application of silkworm in food,but also endangers people’s health.Therefore,it is of great significance to study the types and mechanisms of allergens in silkworm pupae for developing and breeding safe silkworm pupae varieties.This paper aims to screen and develop the low or non-allergic silkworm pupa from the source of germplasm resources.The main research contents are as follows:1.Study on the differences in gene expression of allergens in silkworm pupae.Firstly,about 100 silkworm pupae were collected as test materials.Real Time fluorescence quantitative analysis(qRT-PCR)was used to detect the expression levels of seven major allergens(silkworm hypothetical epidermal protein 30,chymotrypsin inhibitor,small heat shock protein 20.8,vitellogenin,chitinase,30 K family protein,propionate phosphorus)in different silkworm pupa and low allergenic silkworm pupae varieties were preliminarily screened.The results showed that the allergen genes in silkworm pupa of 1007、4034、4020、2016、2030 strains were relatively less.2.Prokaryotic expression of small heat shock protein 20.8(HSP 20.8)and chymotrypsin inhibitor(CI-8A)in silkworm and preparation of antibodies.The HSP 20.8 and CI-8A genes were amplified by reverse transcription-polymerase chain reaction(RT-PCR).The prokaryotic expression vector was constructed by connecting pET-28 a and transferred to E.coli BL21 cells for inducing expression.The results showed that the amplified gene sequence was consistent with the sequence in NCBI database.The prokaryotic expression vectors of HSP 20.8 and CI-8A were successfully constructed.The sizes were about 28 KDa and 47 KDa,respectively,which were in good agreement with the predicted results.At the same time,the antibodies were successfully prepared with titer of 1:51200 and 1:25600.3.Study on the differential expression of allergens in silkworm pupa at protein level.Firstly,the total protein of silkworm pupa was extracted,and the total protein of silkworm pupa was separated by polypropylene gel electrophoresis(SDS-PAGE).After the wet Trarsmembran,the antibody was then reacted with the total protein of the silkworm pupa by immunoblotting(Western-blot),and the content of allergens in silkworm pupae was detected.The results showed that the content of HSP 20.8 and CI-8A in the silkworm pupa of 2025 was the lowest,which can be used as a candidate breeding resource for safe edible silkworm cocoons.