【摘要】 目的　为了探讨 β1 ,4半乳糖基转移酶 Ⅱ和Ⅴ (β1 ,4 galactosyltransferaseI,β 1 ,4 GalT ⅡandⅤ )表达定位 ,本实验通过分子生物学手段 ,制备正、反义 β 1 ,4 GalT Ⅱ和Ⅴ地高辛标记的RNA原位杂交探针。 方法　设计引物 ,提取小鼠脑总RNA ,通过RT PCR方法 ,得到 β 1 ,4 GalT Ⅱ和Ⅴ基因序列 ,将其克隆到pGEM T载体。根据其多克隆酶切位点和Sp6及T7位置 ,分别酶切后作为转录模板 ,通过Sp6及T7RNA聚合酶 ,得到正、反义 β 1 ,4 GalT Ⅱ和Ⅴ地高辛标记的RNA原位杂交探针。检测标记探针的效价后 ,最后通过原位杂交分析标记探针的特异性和杂交效果。结果　本实验得到了高效价的正、反义 β 1 ,4 GalT Ⅱ和Ⅴ地高辛标记的RNA原位杂交探针 ,并表现出很好的杂交效果。结论　正、反义β 1 ,4 GalT Ⅱ和ⅤRNA原位杂交探针的制备 ,为进一步研究 β 1 ,4 GalT Ⅱ和Ⅴ在组织中的表达 ,尤其在神经组织的定位奠定基础更多还原

【Abstract】 Objective In order to study the expression locations of β1,4 galactosyltransferase Ⅱ and Ⅴ(β 1,4 GalT Ⅱ and Ⅴ),the sense and anti sense RNA probes for in situ hybridization of β1,4 galactosyltransferase Ⅱ and Ⅴ were prepared in this study. Method The fragments of β 1,4 GalT Ⅱ and Ⅴ were obtained by RT PCR through total RNA of mouse brains. Amplified cDNA fragment was subcloned into pGEM T Easy. Plasmids were linearized with the restriction enzymes ApaI and SalI. The digoxigenin (DIG) labeled sense and antisense probes were produced using SP6 and T7 RNA polymerase respectively by in vitro transcription according to its protocol. Results Certificated by titer measuring and specificity analysis, the sense and anti sense RNA probes of β 1,4 GalT Ⅱ and Ⅴ were prepared successfully.Conclusion The sense and anti sense RNA probes for in situ hybridization of β 1,4 GalT Ⅱ and Ⅴ were prepared in this experiment, which will provide an approach to study further the locations of β 1,4 GalT Ⅱ and Ⅴ in nerve tissues particularly.更多还原