人钙结合蛋白S100A9基因克隆表达纯化及其对SH-SY5Y细胞作用的研究  

Human Calcium Binding Protein S100A9 Gene Cloning Expression with Purified and Study the Role of SH-SY5Y Cell

作　　者：叶柳[1] 李曌[1] 陈振银[1] 刘笃强 廖键梅 陈雄斌[1] 熊伟[1] 刘兆雨 熊丽荣[1] 李晶[1] 黄琴[1] 李伟[1] 彭彦[1] 唐勇[1] 刘永刚[1] 

机构地区：[1]重庆医科大学基础医学院,组织细胞工程与干细胞研究室,分子医学与肿瘤研究中心,药物化学实验室,重庆400016

出　　处：《基因组学与应用生物学》2016年第12期3260-3267,共8页Genomics and Applied Biology

基　　金：重庆市基础与前沿研究计划项目(cstc2015jcyjA10034)资助

摘　　要：钙结合蛋白S100A9在许多慢性疾病中聚集并可能在一些自身免疫性如老年痴呆中发挥了重要的作用。大量的报道也表明S100A9日益获得人们的注意。通过获取足量重组人S100A9蛋白,探索其对神经母细胞瘤细胞SH-SY5Y的作用及机制。运用全基因合成法合成人S100A9基因,构建人S100A9原核表达重组质粒p ET28a-S100A9,经单酶切法及PCR法鉴定重组质粒已构建成功。利用异丙基硫代-β-D-半乳糖苷(IPTG)在18℃、25℃、37℃分别诱导表达,经聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western blotting鉴定,结果显示18℃时蛋白大量表达于上清中,在37℃时大量表达于包涵体。采用镍亲和层析法分别纯化两种来源重组蛋白,透析后低温冻干获得蛋白粉。使用CCK-8法检测上清来源和包涵体来源的S100A9蛋白对神经母细胞瘤细胞SH-SY5Y的增殖抑制作用。结果显示两种来源重组蛋白对SH-SY5Y细胞具有生长抑制作用,在浓度为0.05 mg/m L时即有明显抑制作用(p<0.01),且二者作用无明显差异(p<0.01)。采用AO/EB双重染色和流式细胞术初步检测S100A9对SH-SY5Y细胞的增殖抑制机理,结果显示该蛋白可促进SH-SY5Y细胞的凋亡。两种来源蛋白之间的比较也显示二者对于SH-SY5Y细胞作用无明显差异(p<0.05)。本研究成功获得足量S100A9重组蛋白,且两种来源S100A9蛋白作用于SH-SY5Y细胞的生物学效应一致。As a calcium binding protein, S 100A9 deposits in many chronic diseases and probably play a crucial role in such as autoimmune diseases, Alzheimer＇s disease etc. Numerous reports show that S100A9 is attracting more and more attention. In order to obtain enough recombinant human S 100A9 （rh-S 100A9） protein and further explore its effects on SH-SY5Y neuroblastoma cells, S 100A9 prokaryotic expression recombinant plasmid pET28a- S100A9 was achieved by using gene synthesis which affirmed built successfully via single enzyme digestion method and PCR. After the induction of isopropylthio-β-D-galactose glucoside （IPTG） in 18℃, 25℃ and 37℃ respectively, olyacrylamide gel electrophoresis （SDS-PAGE） and Western blotting were employed to show the rh- S100A9 was abundantly expressed in the supernatant in 18℃, and in the inclusion body in 37℃. Nickel affinity chromat- ography method was used respectively to purify these rh-S100A9 protein, and then lyophilized after dialysis. CCK-8 proliferation inhibition test was used to detect the effect on SH-SY5Y neuroblastoma cell prolife- ration inhibition of the rh-S100A9 derived both from supernatant and inclusion body. Results showed that the rh-S100A9 protein inhibited the growth of SH-SY5Y cells dramaticly （p 〈0.01） in low concentration （0.05 mg/mL）, but no significant difference between the two sources of rh-S100A9. AO/EB double staining and flow cytometry were used to detect the proliferation inhibition mechanism, results showed that both proteins promote the apoptosis of SHSY5Y cells. Comparison about apoptosis between the two sources of proteins also showed there was no significant difference （p 〈0.05）. Plenty of rh-S 100A9 proteins from supematant and inclusion body obtained and both rh-S 100A9 proteins show consistent biological effects on SH-SY5Y cells.

关 键 词：S100A9 重组质粒 蛋白表达 SH-SY5Y 凋亡 

分 类 号：R346[医药卫生—基础医学]