chOPG RNA探针的制备及其在鸡骨组织的原位杂交

摘要

Objective To get chOPG cRNA probe labeled by digoxigenin. Methods A fragement of chOPG gene was inserted into T vector,and identified with sequencing. Then we linearized the orbicular plasmid with appropriate restriction enzymes, and synthesized the cRNA probe in vitro by SP6 and T7 RNA polymerase. Dot blotting and in situ hybridization of bone were adopted to detect the labeling efficiency. Results chOPG cRNA probe was successfully synthesized. Conclusion Higher sensitivity and creditability chOPG cRNA probe labeled by digoxigenin could be a good tool for further studying the function of OPG.%目的 制备地高辛标记的禽OPG cRNA探针.方法 将目的片段重组到T载体中,构建chOPG/pGM-Teasy重组质粒,EcORI酶切鉴定并测序验证.分别用SalI和NcoI进行酶切得到线性化DNA模板,用Sp6和T7 RNA聚合酶体外转录合成地高辛标记的正反义RNA探针.运用斑点杂交的方法检验探针的标记效率,并通过骨组织原位杂交反应进一步检测探针标记是否制备成功.结果 成功构建出chOPG/pGM-Teasy质粒.结论 获得的高效正、反义chOPGRNA探针,为进一步研究OPG在蛋鸡各组织中的表达奠定基础.