家蚕卵蛋白的 SDS-PAGE 分析(动物科学)

本研究探讨了蛋白质提取方法、十二烷基磺酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)条件对家蚕卵蛋白分离效果的影响。分别用裂解液提取法、TCA沉淀法、冷丙酮沉淀法和TCA-丙酮沉淀法制备蛋白质样品,然后进行6%～12%、6%～15%两种梯度胶的SDS-PAGE,获得了不同提取方法和电泳条件下的电泳图谱用以比较家蚕卵蛋白分离效果。结果表明用TCA-丙酮沉淀法除盐,再用含9 mol.L-1尿素、4%CHAPS、1%DTT的裂解液重悬,最适合家蚕卵蛋白提取;6%～12%梯度胶对家蚕卵蛋白的分离较为适宜。利用这一优化方法对家蚕感染微孢子虫的蚕卵和正常蚕卵进行了比较研究,在分子量66 kD和85 kD处找到了与感染微孢子虫相关的蛋白,为进一步进行正常蚕卵和感染微孢子虫蚕卵的蛋白质组学研究奠定了基础。

SDS-PAGE Analysis of the Eggs of Bombyx mori

The paper discusses the effects of different means to extract the protein of eggs of Bombyx mori as well as to find the best conditions of SDS-PAGE to separate the proteins of eggs of B.mori.Trichloroacetic acid(TCA) precipitation,acetone precipitation and trichloroacetic acid-acetone(TCA-acetone) precipitation were used to extract the protein of eggs of B.mori.The protein was separated by different systems of homogeneous gel in a series of concentrations from 6%(separation gel) ～12%(spacer gel) to 6%(separation gel) ～15%(spacer gel) to search the most appropriate way of extracting the protein of eggs of B.mori and the conditions of electrophoresis.Protein maps of different homogeneous gel concentrations(6%～12%,6%～15%) and different protein extraction methods were obtained.Then the efficiency of the different maps was compared.It showed that removing the salt by TCA-acetone method and dissolving the protein by the lysis buffer consists of urea(9 mol·L-1)、CHAPS(4%)and DTT(1%) were the best way to extract the proteins of eggs of B.mori.Meanwhile the suitable gradient of the homogeneous gel is 6%～12%.The optimized method was explicated to analysis the differences between the eggs of the normal B.mori and whose infected by Nosema bombycis.Finally,the 66 kD protein band and 85 kD protein bands were gained and speculated to associate with the infection of Nosema bombycis.