Preparation,Identification And Allergenicity Assessment Of Tropomyosin From Macrobrachium Nipponense

Macrobrachium nipponense,also called river shrimp,is one of the important freshwater shrimps in China.The shrimp was popular by people because of its delicious and nutritious.However,as one of the 8 major allergens,crustaceans including M.nipponense can induce serious allergic reactions,which caused troubles to the life of people.At present,the consumption of M.nipponense is increasing by years,however,few research reports about allergy of M.nipponense are available compared with other crustaceans.Therefore,the aim of this study is to assess the allergenicity of M.nipponense and to provide theoretical and experimental support for further researching.In this study,the allergenicity of the tropomyosin of M.nipponense was comprehensively evaluated.The research contents are as follows: isolation and identification of M.nipponense tropomyosin;the study of the immune response of tropomyosin;the allergenicity assesssment of tropomyosin used by KU812 cell model and animal model.The main methods,results and conclusions of this study are addressed as follows.1.The tropomyosin was isolated and purified by ammonium sulfate salting out and anion exchange chromatography,respectively.Then,it was identified by performing SDS-PAGE and mass spectrometry.The results showed that the purity of purified tropomyosin was up to 90%.The method is relatively simple,reliable and effective in purification,and it is suitable for small amount preparation of tropomyosin in laboratory.2.The rabbit polyclonal antibody against M.nipponense tropomyosin was obtained by immunizing New Zealand white rabbits with purified tropomyosin as antigen,Indirect ELISA and Western blot were performed for the titer and specificity detection of the polyclonal antibody.The results showed that valence of the prepared rabbit antibody was up to 1:1,638,400,and the specificity of it was strong,which fully met the requirements of subsequent experiments.The high titer and strong specificity of the antibody indicated that the prepared shrimp tropomyosin in this study has good antigenicity.3.The serum of the shrimp allergy patients were incubated with the cells to make the cells sensitized,followed by stimulating the cells by M.nipponense tropomyosin and crude protein to make the cells degranulation.Flow cytometry was used to detectthe expression of intracellular calcium ions,and the release rate of ?-hexosaminidase and the levels of histamine and cytokines in the supernatant were tested in detail.It was shown that the intracellular calcium ion expression of tropomyosin group was79.8%,which was 79.6% higher than that of the control group and 11.4% higher than that of crude protein group;the release rate of ?-aminohexosaminidase from tropomyosin group was 56.88%,which was significantly higher than the other two groups;the contents of histamine,IL-4,IL-6 and TNF-? in the supernatant of the tropomyosin group were 15.47 ?g/ml,3.36 ng/ml,793.33 pg/ml and 411.53 pg/ml,respectively,which were significantly higher than the control group and the crude protein group.It indicated that the river tropomyosin has strong effect on degranulation in KU812 cells,suggesting a strong allergenicity.4.The allergenicity of M.nipponense tropomyosin was evaluated by establishing a Balb/c mouse sensitization model.The results showed that mice in the tropomyosin group developed significant allergic symptoms,and the specific IgE and IgG1 levels in the serum of mice of this group were significantly higher than other groups;the plasma histamine content in the tropomyosin group was 28.11 ?g/mL,which was also significantly higher than other groups;the levels of mMCP-1 in the serum and IL-4,IL-5,IL-13,IL-21,IFN-? that released by spleen cells of tropomyosin group were470.17 ng/mL,24.26 pg/mL,186.40 pg/mL,597.90 pg/mL,1.46 pg/mL,1863.00pg/mL,respectively.The levels of mMCP-1,IL-4,IL-5,IL-13 of tropomyosin group were significantly higher than those of PBS and CT,while the IFN-? level was significantly lower than those in the other groups.Base on the above results,it was found that tropomyosin has strong allergenicity.