Preparation And Functional Analysis Of Polyclonal Antibody Against Syntaxin-1 Protein Of Eriocheir Sinensis

Chinese Mitten Crab(Eriocheir sinensis)is an important economic crab in China.Soluble N-ethyl-maleimide sensitive factor attachment protein receptor(SNARE)is the membrane receptor of N-ethyl-maleimide sensitive factor(NSF)and soluble NSF attachment proteins(SNAP),Syntaxin-1 is one of SNARE proteins.Based on the syntaxin-1 protein,this study provided the basis for exploring the expression and function of SNARE protein family in the spermatogenesis of E.sinensis.The transcriptome information of syntaxin-1 gene of E.sinensis was obtained and analyzed by bioinformatics.The corresponding syntaxin-1 gene was cloned,and the recombinant cloning vector was transferred into DH5? cultured in vitro.The results showed that the syntaxin-1 protein of E.sinensis was a hydrophilic unstable protein with a transmembrane region and no signal peptide.It was a mature protein.The syntaxin-1 protein of E.sinensis had homology with syntaxin-1a protein of American crab.The size of syntaxin-1 gene cloned was 250-500 bp,which was consistent with the theoretical value(397 bp).The corresponding expression vector syntaxin-1-p ET-30 a was constructed and transformed into DE3 cultured in vitro.Syntaxin-1 protein was purified by histidine tag on pet-30 a.It was found that the purified protein was 20-31 kd,which was consistent with the theoretical value(21 kd).The purified syntaxin-1 protein was used as immunogen to prepare polyclonal antibody,and the titer of polyclonal antibody was detected.The titer of polyclonal antibody was1:25600 and more than 1:10000,which met the requirements of antibody preparation.By using the polyclonal antiserum as the first antibody,the localization of syntaxin-1protein in various spermatogenic cells of E.sinensis was analyzed by immunofluorescence and immunohistochemistry.The results of immunofluorescence showed that syntaxin-1protein was located on the cell membrane in spermatogonia,primary spermatocytes and secondary spermatocytes,but in mature spermatocytes it was located on the nucleus.The secretion of syntaxin-1 in spermatogonia,primary spermatocytes and secondary spermatocytes increased in turn.The staining effect of multi antibody group was significantly better than that of antibody group.At the same time,immunohistochemical results also showed that the localization of the protein was consistent with immunofluorescence results.In conclusion,syntaxin-1 gene was successfully cloned,and syntaxin-1-p ET-30 a expression vector was constructed.Syntaxin-1 protein was successfully synthesized and purified.The purified syntaxin-1 protein was used as immunogen to prepare polyclonal antibody with satisfactory titer.Immunofluorescence and immunohistochemistry showed that syntaxin-1 protein was located on the cell membrane in spermatogonia,primary spermatocytes and secondary spermatocytes,and on the nucleus in mature spermatozoa.Based on the results of immunofluorescence and immunohistochemistry,it can be inferred that syntaxin-1 protein mainly plays a role in the late stage of spermatogenesis,and its main function is to complete sperm acrosome reaction.After the formation of mature sperm,SNARE complex disintegrates and syntaxin-1 protein is transferred into the nucleus.