Novel polypeptide
a polypeptide and polypeptide technology, applied in the field of new polypeptides, can solve the problems of difficult to synthesize sialyl lewis x sugar chain with 2,3-siaryltransferase, unknown enzyme, and above enzymes that have not been identified, and achieve the effect of efficiently secreting and efficiently secreting
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example 1
Cloning of a Novel Mouse α1,3-Fucosyltransferase Gene (cDNA)
(1) Preparation of mRNA from Mouse Brain
[0341] About 30 μg of mRNA was prepared from BALB / c mouse brain using a mRNA extractor kit, Oligotex™-dT30 (manufactured by Roche). Reagents and methods were employed as described in the instructions attached to the kit.
(2) Construction of a Mouse Brain cDNA Library
[0342] Using 8 μg of mRNA obtained from the mouse brain in (1) above and a kit (SUPERSCRIPT Choice System for cDNA Synthesis manufactured by GIBCO BRL), double-stranded cDNAs were synthesized with oligo-dT as primers.
[0343] SfiI linkers were ligated to the both ends of the double-stranded cDNAs as described below.
[Addition of Sfi I Linker]
[0344] A single-stranded DNA represented by SEQ ID NO:6 and a single stranded DNA represented by SEQ ID NO:7 were synthesized using a 380A DNA synthesizer (manufactured by Applied Biosystems).
[0345] Each of the synthesized single-stranded DNAs 50 μg was independently dissolved in...
example 2
Synthesis of the Lewis x Sugar Chain and the Lewis y Sugar Chain in a Human Culture Cell into which a Mouse Fuc-TIX Expression Plasmid Obtained in Example 1 has been Introduced
[0390] A control plasmid (pAMo) and a mouse Fuc-TIX expression plasmid (pAMo-mFT9) were separately dissolved in TE buffer to 1 μg / μl, introduced into Namalwa cells by electroporation method, thereby obtaining transformed cells.
[0391] The transformed cells were subjected to indirect fluorescent antibody staining using an anti-Lewis x sugar chain antibodies (PM-81 or 73-30), an anti-Lewis y sugar chain antibody (AH-6), and an anti-sialyl Lewis x sugar chain antibody (KM93).
[0392] Indirect fluorescent antibody staining was performed according to the methods described in Example 1 (3). When compared to the cells into which pAMo had been introduced, the cells into which pAMo-mFT9 had been introduced showed an approximately 20-fold increase in reactivity (peak value of histogram) against each of the anti-Lewis x ...
example 3
In Vitro Substrate Specificity of Mouse Fuc-TIX
[0402] In vitro substrate specificity of Fuc-TIX encoded by the mouse Fuc-TIX cDNA obtained in Example 1 was examined as follows.
[0403] To express the mouse Fuc-TIX in animal cells, the mouse Fuc-TIX cDNA was subcloned into an expression vector pCDM8 for animal cells as described below.
[0404] That is, a 1.2-kb HindIII-AseI fragment containing an open reading frame (ORF) of the mouse Fuc-TIX cDNA was isolated from pAMo-mFT9. The cut ends were blunt-ended with T4 DNA polymerase, then subcloned into an EcoRV site of pBluescript SK(−).
[0405] A plasmid, in which the cDNA was subcloned in a direction such that the 5′ and 3′ sides of the cDNA was present near HindIII and PstI sites of the cloning sites, respectively, was selected. The plasmid was excised with HindIII and PstI to isolate a 1.2-kb fragment containing the mouse Fuc-TIX cDNA.
[0406] The 1.2-kb fragment was subcloned between HindIII and PstI of pCDM8 to construct a mouse Fuc-TI...
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