【作者】 麻晓庆；

【导师】 李庆伟；

【作者基本信息】 辽宁师范大学 ， 细胞生物学， 2007， 硕士

【摘要】 Grimin为源自于日本七鳃鳗口腔腺的GRIM—l9样抗肿瘤基因重组蛋白。GRIM-19是新近发现的一种”细胞死亡激活因子”,由于GRIM-19能特异性抑铝STAT3(Signal transduceran activat or of transcription,信号转导与转录活化因子)分子发挥作用来阻断肿瘤细胞的信号传递途径,促进肿瘤细胞凋亡或者逆转恶性表现型,而日本七鳃鳗Grimin与GRIM-19具有相同的功能结构域及较高的序列同源性,这使得Grimin有望成为高效低毒、副作用小的抗肿瘤基因工程新药。鉴于此,本论文从日本七鳃鳗中提取GRIM-19的同功能基因,将其命名为Grimin,并对其进行了初步的研究和验证。本课题组已先期完成七鳃鳗口腔腺cDNA文库的构建,所构建的库容量为2.1×1O。pfu·ml-1。随机挑选单克隆单向测序后,共得到1281条有效EST序列。对其进行生物信息学分析后,发现了1条能翻译出GRIM-19结构域的EST序列。对此段mRNA进行全长钓取、开放阅读框寻找、测序及b1asting同源性比较后,发现此一功能基因与非洲蟾蜍GRIM-19的同源性为66%,与河豚GRIM-19的同源性为6896。根据其设计引物进行RT—PCR,获得了Grimin的429bp的功能基因。进一步将此功能基因构建入表达载体pET23b,并转化入克隆菌DH5a后进行阳性转化子的筛选鉴定。本论文主要将带有组氨酸标签的pET23b—Grimin重组质粒从克隆菌中提取出,然后CaCl2法转化入表达菌E.coliRosetta中进行IPTG诱导表达;经组氨酸亲和层析柱对表达的重组蛋白进行纯化。表达产物为包涵体,进行包涵体透析复性后得到有生物活性的蛋白,将其命名为Grimin蛋白。获得了具有生物活性的Grimin蛋白之后,本论文进一步进行了对Grimin的生物学活性的检测。培养人脐静脉内皮细胞ECV304及人白血病细胞HL60,收集细胞,利用MTT比色法,双苯并咪唑(Hoechst33258,H0)染色及流式细胞仪分析等实验证明,Grimin蛋白具有抑制肿瘤细胞生长并促进其发生凋亡的生物学活性;而且本课题对Grimin进行了质脂体包被的小规模实验,以使包被Grimin蛋白透过细胞膜质脂双分子层而达到细胞内起作用,本实验是以未加蛋白作用的细胞和未加蛋白仅加脂质体的细胞分别作对照;加入蛋白和脂质体混合物的结果与对照相比;结果显示经过脂质体包裹的蛋白作用的细胞出现明显的细胞凋亡,并且脂质体对细胞没有任何刺激作用,从而证明了可以利用脂质体包被作为药物剂型在抗肿瘤药物中加以应用。总之,本论文为了研究GRIM-19诱导肿瘤细胞凋亡方面的作用,成功地通过基因工程手段从日本七鳃鳗口腔腺中获取了新的功能基因以及蛋白,并较为系统地对Grimin的相关生物学活性进行了初步研究。这些实验结果都提示了Grimin作为一种肿瘤细胞死亡催化剂的潜在应用价值。更多还原

【Abstract】 Grimin roots in the anti-tumor gene recombination protein GRIM-19 of Lampetra japonica buccal gland, where GRIM-19 is a kind of the cell death activation factor which was recently discovered. Because the tumor cell transmission can be blocked by the GRIM-19’s specific suppressor to the rule of the STAT3 which stimulate the tumor cell apoptosis or inverts malignant phenotype, and the Grimin of Lampetra japonica has the same functional domain and the higher sequence homology with GRIM-19. So it hopefully becomes the anti-tumor genetic engineering new medicine with powerful effects, little poison and little negative factor.In view of it, we has withdrawn successfully the gene with the same function as GRIM-19 from Lampetra japonica. It has been conducted the preliminary research and the corresponding confirmation,named as Grimin.Our team have finished the constructing work of the gene library about Lampetra japonica buccal gland’s cDNA with its 2.1×10⁶pfu·ml^-1 capacity. We have withdrawn the total RNA from the Lampetra japonica in its migration to Heilongjiang basin, using our independently design premier to carry on the RT-PCR expanding, and have obtained the new function gene of 429bp long Grimin. The premier design is basised on the GRIM-19 homology protein sequence of disintegrins published on NCBI, which deduces the DNA sequences and designs two oligonucleotide primers. the result shows, this function gene and the African toad GRIM19 homology is 66%, and the globefish GRIM19 homology is 68%.The paper follows the above works , and the histidine labeled pET23b-Grimin has been transformed into expresses in fungus E.coliRosetta to carry on the IPTG primer expression; after the affinity column analysis of histidine to purify the expressed reorganization protein. The active protein is obtained after denaturation and renaturation of its product inclusionbody, and named Grimin.obtained the biological activity Grimin protein, we have further carried on the partial biological activity examination to it. Through Raising personal vena umbilicus endothelial cell ECV304 and personal leukemia cell HL60, collecting cell , using the MTT color method, the double benziminazole （Hoechst 33,258, Ho） dyes and flows experiment analysis, the Grimin protein has been proved its biology activeness which can suppress tumor cell grows and promote it apoptosis; Moreover we has carried on the little experiment of the lipide body peridium about Grimin, which enables the Grimin peridium protein penetration cell membrane to function in the cell. The experiment make the separately comparison with the cell added protein and the cell not added the protein only plus lipide cell; the results show the cells with lipide protein wraped appear the obvious perish and lipide body does not enduce any stimulation to cell. Thus it proves that the lipide body peridium may be taken as the anti-tumor medicine dosage to apply.In the paper we successfully obtain the new functional gene and protein from Lampetra japonica through the genetic engineering method to study the GRIM-19 induce tumor cell apoptosis, and make some preliminary research to the relative biology activity about Grimin. These experimental results can prompt Grimin the latent application value to be taken as one kind of catalyst inducing tumor cell to death.更多还原