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为了制备小鼠 β-1,4-半乳糖基转移酶 ( β-1,4-galactosyltransferase ,β-1,4-Gal T- )地高辛标记的 RNA探针以探讨β-1,4-Gal T- m RNA在坐骨神经组织的表达定位 ,本研究用提取的小鼠脑总 RNA,采用 RT-PCR方法 ,得到β-1,4-Gal T- 的 DNA片断 ,将其克隆到 p GEM-T载体 ;采用体外转录的方法合成地高辛标记的正、反义β-1,4-Gal T- RNA探针 ;运用点杂交的方法分析标记探针的灵敏度 ;最后应用该探针 ,通过原位杂交的方法 ,分析 β-1,4-Gal T- m RNA在小鼠坐骨神经的表达。结果 :构建了β-1,4-Gal T- /p GEM-T质粒 ,获得了高效价的地高辛标记的正、反义β-1,4-Gal T- RNA探针 ,应用该探针发现 β-1,4-Gal T- 在小鼠坐骨神经髓鞘中有表达。以上结果表明 ,制备的地高辛标记的正、反义 β-1,4-Gal T- RNA原位杂交探针可特异地检测β-1,4-Gal T- m RNA在组织中的表达。本研究为进一步分析β-1,4-Gal T- 在坐骨神经及其它神经组织发育和损伤过程中的表达奠定基础。
In order to prepare the digoxigenin labeled β-1,4-galactosyltransferase (β-1,4-galactosyltransferase) Gal-T-RNA was localized in the sciatic nerve tissue. In this study, total RNA was extracted from mouse brain by RT-PCR to obtain β-1,4-Gal T- T vector; digoxin-labeled positive and antisense β-1,4-Gal T-RNA probe was synthesized by in vitro transcription; the sensitivity of labeled probe was analyzed by dot hybridization; finally, In situ hybridization was used to analyze the expression of β-1,4-Gal T-m RNA in the sciatic nerve of mice. RESULTS: The β-1,4-Gal T- / p GEM-T plasmid was constructed and a high titer digoxigenin labeled positive and antisense β-1,4-Gal T-RNA probe was obtained. It was found that β-1,4-Gal-T was expressed in the myelinated sciatic nerve of mice. The above results showed that the prepared digoxigenin-labeled antisense β-1,4-Gal T-RNA in situ hybridization probe can specifically detect the expression of β-1,4-Gal T-RNA in tissues . This study laid the foundation for further analysis of the expression of β-1,4-Gal-T in the development and injury of sciatic nerve and other nervous tissues.