Identification And Function Of Tetrodotoxin-binding Protein In Takifugu Bimaculatus

In this paper,the distribution and effects of tetrodotoxin-binding protein in pufferfish were investigated based on the surface plasmon resonance（SPR）technique,and the mechanism of tetrodotoxin transfer and enrichment in the body of pufferfish was investigated.The results of the study are as follows:1.Fishing and molecular docking fitting of tetrodotoxin（TTX）-binding proteinsBased on SPR technology,TTX-related binding proteins were fished from the ovaries of wild Takifugu bimaculatus,and three proteins were screened by mass spectrometry:TbRACK1,TbSERBP1 and Tbprx-1.The 3D model structures of the three proteins,TbRACK1,TbSERBP1 and Tbprx-1,and their binding modes to TTX were predicted by SWISS-MODEL and Schrodinger software.The results showed that all three proteins could bind TTX with binding free energies of-6.82 kcal/mol,-7.48 kcal/mol and-5.81 kcal/mol,respectively.2.Functional identification of TTX-binding proteinThe recombinant proteins of rTbRACK1,rTbSERBP1 and rTbprx-1 were expressed in the prokaryotic nucleus.The equilibrium dissociation constants（KD）of the three proteins with TTX were verified by SPR as 2.5×10^-3 M,6.3×10^-4 M and 4.1×10^-5 M,respectively,indicating that all three proteins could bind TTX.3.Expression profiles of TTX-binding protein genes and after gavage of TTXThe tissue distribution of TbRACK1,TbSERBP1 and Tbprx-1 in the one-year old and two-year old farmed Takifugu bimaculatus,as well as the spatial and temporal distribution in the liver and ovaries of Takifugu bimaculatus after gavage of TTX were examined by qPCR.The results showed that the m RNA of TbRACK1 was mainly distributed in the heart,gonad,spleen and gill,the m RNA of TbSERBP1 was mainly distributed in the gonad and kidney,and the m RNA of Tbprx-1 was mainly distributed in the kidney,intestine and gill in the one-year old Takifugu bimaculatus.The m RNAs of TbRACK1,TbSERBP1 and Tbprx-1 were all highly expressed in the gill of the two-year old Takifugu bimaculatus,and Tbprx-1 was also highly expressed in the kidney.The expression characteristics of each gene in liver and ovary at different time points were detected by qPCR afterTTX gavage in cultured Takifugu bimaculatus.4.Functional study of rTbprx-1 activityA rabbit polyclonal antibody to rTbprx-1 was prepared,and the binding ability of rTbprx-1 to TTX was verified using ELISA to obtain an equilibrium dissociation constant of 1.36×10^-6 M.The in vivo action of TTX in mice under the action of rTbprx-1 was photographed using live imaging,and the results indicated that rTbprx-1 may be involved in the transport of TTX in mice;meanwhile rTbprx-1 has the function of promoting the transport of TTX into cells.