摘要
脓毒症(sepsis)是多器官功能障碍(MODS)的前兆,是当前危重病医学面临的难题之一,它也是导致严重烧伤、创伤患者死亡的主要原因之一;其发病机制与内毒素(LPS)激活血管内皮细胞诱发并加重全身炎症反应密切相关。脓毒症的病理生理机制复杂,现行的治疗措施有限,因此我们深入研究其发病机制就显得尤为重要。
近年来,由于血管内皮细胞是内毒素作用的直接靶细胞之一,国内外对LPS激活内皮细胞的作用途径、特异受体、胞内信号转导等方面研究较多。本研究室前期应用抑制消减杂交(SSH)和cDNA末端扩增技术(RACE),从LPS刺激后的人脐静脉内皮细胞中克隆到一个人类新基因全长cDNA序列,经Northern blot验证后作为人类新基因被GenBank全长序列库所接受(No.AY074889)。因其在受LPS刺激的内皮细胞中上调表达,故暂时命名为内皮高表达脂多糖相关因子1(endothelial-overexpressed lipopolysaccharide-associated factor 1,EOLA1)。
目前,国外尚无关于该基因的相关研究,本实验室对EOLA1基因做了一些前期研究工作,并经生物信息学分析显示:该基因全长1404个bp,其定位于染色体Xq27.4,编码蛋白由158个氨基酸组成,分子量17.89kDa,等电点6.43,亲水性好,为可溶性蛋白。其二级结构含α螺旋、β片层结构和β转角,并形成一个螺旋—转角—螺旋(HTH)基序。EOLA1氨基酸序列包含N-糖基化位点、PKC磷酸化位点和酪氨酸激酶2磷酸化位点。
多组织Northern blot显示EOLA1基因除在LPS刺激的内皮细胞上调表达外,在骨胳肌、心脏、肾脏、肝脏和胎盘有较强的表达,在结肠、小肠、脾脏有较弱的表达,而在脑、胸腺、肺和外周白细胞则未见表达。在7种癌细胞株上,显示在早幼粒白血病系HL-60、海拉细胞系S3、慢性骨髓性白血病系K-562和肺癌细胞系A549都有表达,而在成淋巴细胞瘤性白血病MOLT-4和结直肠腺癌SW480表达水平最高,但在黑素瘤G-361无表达。在经过G418压力筛选后的稳定表达EGFP-EOLA1(enhanced green fluorescent protein-EOLA1)融合蛋白的ECV304细胞株中发现EOLA1蛋白为全细胞分布。酵母双杂交显示EOLA1蛋白与细胞内金属硫蛋白相互作用,可能参与细
Sepsis is an auspice of multiple organ dysfunctional syndrome (MODS) and an intractable problem, which is facing with in critical and serious illness medicine. Sepsis is also the one of main causes of death in serious burn and traumatic patients. EC is one kind of the main target cells that are directly activated by LPS, and effect on EC by LPS with regard to signal transmitting and special receptor and route of action has been studied more. The EOLA1 gene (a human novel gene, GenBank Accession No. AY074889) was cloned in our laboratory by the effective technique of mRNA differential display (comparing the differential gene expression between normal ECV304 and treated by LPS). Using the SMART RACE technique, the full-length cDNA of the EOLA1 gene was cloned. It contains 1404 nucleotides, 474 nucleotides of the open reading frame that predicts 158 amino acids. The genomic DNA contains 5 exons, spans about 6294 bps, and is mapped to human chromosome Xq27.4. Analyzing by bioinformatics methods found that EOLA1 secondary structure contains α -helix, β -Lamellosa and β -turn, and a helix-turn-helix (HTH) motif. This information indicated that EOLAl may be as a transcription factor, which play an important role in process of activating Human EC.Northern blot with some kinds of Human tissues and carcinoma cells revealed an extended expression profile of EOLA1. It was highly expressed in skeletal muscle, liver, placenta and lowly expressed in the spleen, heart and no expressed in brain, colon, small intestine, thymus, lung and peripheral blood leukocyte; The yeast two-hybrid experiment has showed the interaction of EOLA1 and MT2A which may play some roles of apoptosis and growth and anti-inflammation in human EC. Now the biological function of EOAL1 is unknown, so preparing anti-EOLA1 antibody is very important to functional studying of EOLA1 in the future.The open reading frame of EOLA1 gene was cloned by RT-PCR from human ECV304 cells, and was judged as 500bp length by agarose gel electrophoresis. Recombinant
plasmid was successfully constructed and identified by colony PCR and sequence measurement. Recombinant EOLAl protein was successfully expressed in BL21(DE3)plysS and the expressed protein in E.coli mainly exist in inclusion body form. The molecular weight of EOLAl is about 20x103 by SDS-PAGE analysing and Western blot. The optimization of induction conditions which include Incubate at 37°C for 4h, E.coli BL21(DE3)plysS, lmmol/L IPTG and 100 u g/ml ampicillin was founded through experiment. The target protein has been expressed in large-scale under above induction conditions: 9.6g of the cells collected by centrifugation from induced 2000ml culture and 0.76g of inclusion bodies were collected after primary purification including destruction of bacteria, washing-up and isolation. 100ml of target protein solution with 124.16ug/ml concentration was obtained after immobilized metal affinity chromatography and renaturation. Peptide mass fingerprinting show the matched peptides cover 32% (56/172 AA's) of the theoretic EOLAl protein. The anti-EOLAl polyclonal antibody was collected through BALB/c rat immunization and identified by Western-blot. ELISA showed its efficiency has arrived at 1:10000.In conclusion, the opening read frame of EOLAl has been cloned by RT-PCR from ECV304 cells and identified by sequence analysis, which further confirms the veracity and facticity of EOLAl. EOLAl recombinant protein can be expressed in E.coli largely in inclusion body form. Anti-EOLAl polyclonal antibody that is produced by our experiment can detect expression of nature EOLAl in ECV304 cells.
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