抑癌基因PTEN／MMAC1的cDNA克隆及其表达质粒的构建

为了获得了PTEN/MMAC1的cDNA并构建其酵母双杂交系统中的诱饵质粒和逆转录病毒表达质粒。利用RTPCR方法，从293细胞中扩增出一约12.kb的DNA片段，与pGEMT Easy连接，作全自动测序确证，重组入pLexA载体，构建成pLexA-PTEN/MMAC1，并用醋酸锂法转化酶母菌EGY48（p8op-LacZ），在选择性培养基上观察pLexA-PTEN/MMAC1在EGY48(p8op-LacZ）中的表达情况；同时，PTEN/MMAC1的cDNA也重组入pLXSN构建pLXSN-PTEN/MMAC1。结果PCR获得1.2kb的DNA序列与献报道的PTEN/MMAC1的cDNA一致，转化的酵母菌在选择性培养上培养3d后，长出约1mm大小的白色菌落；pLXSN-PTEN/MMAC1可酶切出1.2kb的PTEN/MMAC1片段。结果表明获得了PTEN/MMAC1的cDNA，pLexA-PTEN/MMAC1可作为酵母双杂交系统中的诱饵质粒，而pLXSN-PTEN/MMAC1的构建为进一步研究其抑癌作用打下了基础。

Cloning of the PTEN/MMAC1 cDNA and construction of its expression plasmid

In order to obtain PTEN/MMAC1 cDNA and construct the bait plasmid of yeast two?hybrid system and its retroviral plasmid. About 1.2 kb DNA fragment was amplified from the 293 cell by RT?PCR. After automatic sequenced, the fragment was ligased with the vector pLexA to construct pLexA?PTEN/MMAC1. The recombinant plasmid was transformed into yeast EGY48(p8op?LacZ), and the expression of pLexA?PTEN/MMAC1 in the yeast was observed. Meanwhile, PTEN/MMAC1 cDNA fragment was ligased with retroviral vector pLXSN to construct pLXSNPTEN/MMAC1. Results is that the cDNA fragment by RT?PCR was same with reported PTEN/MMAC1 cDNA, and about 1 mm white yeast clone transformed with pLexA?PTEN/MMAC1 was grown in the selective medium after 3 days. About 1.2 kb DNA fragment was shown from the digested pLXSN?PTEN/MMAC1. Results indicated that the PTEN/MMAC1 cDNA has been cloned. pLexA?PTEN/MMAC1 can be used as a bait plasmid of yeast two?hybrid system. While the plasmid pLXSN?PTEN/MMAC1 make the base for further studying the tumor suppression function of PTEN/MMAC1.