高迁移率族蛋白B1基因的克隆及其与神经管发育的相关关系

目的:构建神经管cDNA文库,寻找神经管发育相关基因。方法:提取E8.5 d金黄地鼠神经管总RNA;SMART技术构建神经管cDNA噬菌体表达文库;重组噬菌体PCR鉴定。将出现频率很高的重组噬菌斑,经质粒转化、酶切鉴定和DNA序列分析,证实为高迁移率族蛋白B1基因(HMGB1)。将HMGB1 cDNA片段回收、纯化,制备探针;Northern杂交检测不同发育阶段神经管中HMGB1 mRNA的表达变化。结果:构建的HMGB1cDNA片段含有完整的cDNA序列。Northern杂交显示:随胚胎发育,神经管HMGB1 mRNA表达量逐渐增加,E10 d增加最为明显,E12 d仍处于较高水平;而8.5 d高温致畸胚神经管,HMGB1 mRNA表达量较对照组明显减少。结论:HMGB1基因的表达与神经管发育及高温致神经管畸形的发生密切相关。

Cloning of high mobility group box B1 gene and its relationship with development of neural tube

Objective: To construct the neural tube cDNA library and to investigate the genes associated with the development of the neural tube.Methods: Total RNAs were extracted from E8.5d neural tube of golden hamster using TRIzol reagent;the neural tube cDNA library was constructed by SMART method.By PCR screening of recombinant cDNAs from the constructed library,we obtained a recombinant plaque with high frequence. After insertion into plasmid,enzyme identification and sequence analysis,the cDNA fragment was confirmed to be high mobility group box B1 gene(HMGB1).Then the HMGB1 cDNA was extracted,purified and labelled.Total RNAs of nerual tube at each time during neurulation were isolated respectively and the level of HMGB1 mRNA in the neural tube of each group were detected by Northern blot.Results: The length of the cDNA was 1()242()bp containing full ORF.Northern blot showed that the level of HMGB1 mRNA was increased gradually during neurulation,and increased significantly at E10 d,and also high in the neural tube at E11()d and E12()d,whereas decreased at the 8.5 d neural tube of the neural tube defects embryos.Conclusion: HMGBl gene is closely correlated with the development of the neural tube and neural tube defects caused by hyperthermia.