The serine/threonine protein kinase B(PKB) is related to cellular survival and regulation. PKB is composed of PH domain, catalytic domain and carboxyl terminal regulator domain. The PH domain of PKB is crucial to the activation of kinase. In order to investigate the function and the structure function relationship of PKB, the cDNA coding fragment of PKB PH domain was amplified from human dental pulp mRNA by RT PCR and cloned into pMD18 T vector to analyze the sequence. The result showed that DNA sequence of cloned human PKB PH domain was consistent with that reported previously. To express PKB PH domain, the cDNA was subcloned into expression vector pRSET A which was then transformed into E.coli BL21(DE3) pLysS, and the strain highly expressing soluble 6His PKB PH domain in minimal medium was obtained. The fusion protein was purified by Ni 2+ NTA agarose beads. The secondary structure of the purified 6His PKB PH domain fusion protein was analysed by circular dichroism. The results indicated that the PH domain was composed of α helix 1 7%,β pleated sheet 80 5% and radom coli 17 8%.
目的:为探讨SPARC(secreted protein acidic and rich in cysteine)在人恶性肿瘤发生、发展中的作用及其分子机制,进一步明确SPARC发挥作用的方式及其与肿瘤发生类型的关系。方法:我们首先提取了人乳腺癌细胞系MCF-7的总RNA,在对总RNA进行纯度与定量检测后,利用RT-PCR的方法,以该总RNA为模板,将其反转录为cDNA;再设计引物,以该cDNA为模板,利用PCR扩增出包含Sparc编码区的DNA片段,将该产物纯化后通过T-A克隆连接入pMD20-T载体,利用菌落PCR及DNA测序进行鉴定。以pMD20-T-Sparc为模板,我们设计了特异的针对Sparc全长编码区的引物,并在引物5'端分别加入BamHI、HindIII酶切位点,通过PCR将Sparc编码区扩增出来,经纯化及双酶切后与真核表达载体pcDNA3.1myc-his(-)相连,再经菌落PCR和DNA测序进行鉴定。通过瞬时转染的方法,利用脂质体将所构建的重组SPARC真核表达载体转染HEK293细胞,48h后裂解所培养的细胞,使用western blot检测有无SPARC的表达。结果:测序证实所克隆的Sparc编码区cDNA正确地插入pcDNA3.1myc-his(-)中,western blot检测证实其在HEK293细胞中得到表达,而空载体转染的细胞则无表达,说明所构建的pcDNA3.1myc-his(-)-Sparc能够成功表达。结论:我们成功克隆了人Sparc cDNA,构建了其真核表达载体,并在HEK293细胞中得到有效表达,从而为进一步研究人SPARC的功能及其与肿瘤的关系奠定了基础。
