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Sugar sphingolipids Gb3 (Globotriaosylceramide) widely present in mammalian cell surface, in cell growth, differentiation, recognition, signal transduction plays a very important role. Gb3 on the cell membrane is the Shiga toxin (Shiga toxin, STx) receptor, and Shigella cause toxic diarrhea, hemorrhagic colitis, hemolytic uremic closely related. STx by Shiga toxin Shigella dysentery (Shigella dysenteriae) has emerged as an important cause viral virulence factors, having enzymatic activity of the A subunit (STxA) and the role of receptor-binding B subunit (STxB) two parts. Studies show that, in mammalian cells, STxB and Gb3 binding, by the early endosomal / intracellular loop directly to the Golgi body, and then the reverse transport to the endoplasmic reticulum, rather than through the late endosomal degradation of the environment. However, another study showed that the fluorescently labeled cells Gb3 join, some Gb3 also reached the Golgi apparatus. However, some of the existing evidence, Gb3 and its ligands STxB endocytosis may take a different approach: cell intrinsic pathway and by the Gb3 ligand STxB induced STxB way. In this paper, HeLa cells were used as experimental materials, from two aspects of the ligand structure and ligand endocytosis pathway relations. The first part, constructed by molecular cloning of three unequal strength and Gb3 binding mutant STxB, a study comparing wild type and mutant ligand ligand intracellular transport. The second part compares the different ligands (natural and artificial ligands STxB antibody ligand Gb3 38-13) endocytosis pathway, and clarify the STxB transport path in the cell is induced by the STxB. In the first part, first summarize the literature to determine construct three mutants were W34A, D17E and F30A,. In combining ability, W34A strongest, D17E centered, F30A weakest. PSU108 plasmid as a template, mutagenesis by overlap extension PCR amplification of a cDNA fragment of the mutant. PSU108 mutant cDNA and vector were used to Sph I, Not I enzyme double cutting, and then with T4 DNA ligase gene cloned. Recombinant plasmid was transformed into E. coli DH5α, positive clones by restriction enzyme digestion of plasmid little mention is correct, were sequenced. The sequencing results consistent with the experimental design. Correct sequencing of the bacteria strains and expression STxB enlarged cultured at 42 ℃ under the conditions of induction of recombinant proteins and the wild-type protein. Osmotic shock method to extract a periplasmic protein, SDS-PAGE electrophoresis with positive protein bands appear. Periplasmic extract the first through the Q-Sepharose FF ion-exchange column preliminarily purified, and then MonoQ HR 5/5 strong anion exchanger further purification. SDS-PAGE and identification of the target protein purity> 95%, Western Blot test for the intended purpose of pure protein protein. Finally, the quantitative Coomassie protein. In HeLa cells for the study of systems, immunofluorescence and compared STxB, W34A, D17E, F30A transit situation. The results showed that, after 18 ℃ cultured 2 h, these four proteins can be gathered in the early endosomes / recycling endosomes. After 37 ℃ for 3 h, these four proteins can be targeted Golgi apparatus. But part of the rendered STxB endoplasmic reticulum localization characteristics, suggesting that the weakening of the receptor binding effects from the early endosomes / loop endosomes to the Golgi transport speed. After 37 ℃ for 4 h, STxB showed typical endoplasmic reticulum localization characteristics, the mutant ligand also Golgi, and appears F30A slowest. After 37 ℃ for 16 h, STxB endoplasmic reticulum localization characteristic is typical, and the mutant ligand still Golgi. Further studies have shown that mutants unable to reach the endoplasmic reticulum, Golgi degradation after arrival. Indicate the strength of influence receptor binding to the endoplasmic reticulum transport, namely STxB intracellular transport depends on STxB-Gb3 complexes stabilizing effect. In the second part, compared STxB and Gb3 antibody 38-13 in HeLa cells endocytic pathway. STxB is the naturally occurring ligands Gb3, and 38-13 can be seen as Gb3 antibodies synthetic ligands Both ligands are binding oligosaccharide portion Gb3. Based on the results, Gb3 antibody 38-13 binds to the cell surface, but can not enter the cell, and STxB reverse transport path is entirely inconsistent. Description STxB transport path in the cell is induced STxB, and not inherent in the use of the cell transfer route Gb3.
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