Cheng Jun, Wang Lin, Li Ke, Lu Yin-Ying, Liu Yan, Duan Hui-Juan, Hong Yuan, Wang Gang, Li Li, Zhang Ling-Xia
Gene Therapy Research Center, Institute of Infectious Diseases, 302 Hospital of PLA, Beijing 100039, China.
Hepatobiliary Pancreat Dis Int. 2002 Feb;1(1):87-91.
To study the function of augmenter of liver regeneration (ALR) as a regulatory factor that specifically stimulates hepatic cell regeneration, we constructed yeast expressive vector of ALR and expressed it in yeast cells.
Total RNA was extracted from HepG2 cells, and reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the coding region of ALR. The products were cloned into PGEM-T vector and sequenced, then cloned into PGBK T7 vector. The recombinant plasmid PGBK T7-ALR was transformed into yeast AH109. The yeast protein was extracted and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization technique.
DNA sequencing results confirmed that the coding region of ALR was correctly inserted into the yeast expression vector, and Western blotting assay showed that recombinant ALR was successfully expressed in yeast. Its molecular weight was identical to the theoretical value of 15 000 Da; the protein was found inside the yeast cells.
The successful expression of ALR in yeast cells makes it possible to study further on its biological function.
为研究肝再生增强因子（ALR）作为特异性刺激肝细胞再生的调节因子的功能，我们构建了ALR的酵母表达载体并在酵母细胞中进行表达。
从HepG2细胞中提取总RNA，通过逆转录聚合酶链反应（RT-PCR）扩增ALR的编码区。产物克隆到PGEM-T载体并测序，然后克隆到PGBK T7载体。将重组质粒PGBK T7-ALR转化到酵母AH109中。提取酵母蛋白，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质印迹杂交技术进行分析。
DNA测序结果证实ALR的编码区正确插入酵母表达载体，蛋白质印迹分析表明重组ALR在酵母中成功表达。其分子量与理论值15 000 Da相同；该蛋白在酵母细胞内被发现。
ALR在酵母细胞中的成功表达使得进一步研究其生物学功能成为可能。
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