發(fā)布時(shí)間：2018-01-17 21:02

  本文關(guān)鍵詞：人源胸腺素α原和白介素2融合基因的真核表達(dá)及免疫保護(hù)效果初探　出處：《廣西師范大學(xué)》2008年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 胸腺素α原 白介素2 融合基因 真核表達(dá)

【摘要】： 人源胸腺素α原(prothymosinα,proTα)是具有促進(jìn)細(xì)胞增殖和顯著的免疫刺激活性的酸性蛋白,主要用于治療腫瘤、肝炎、免疫系統(tǒng)等疾病。白介素2(interleukin 2,IL-2)是一種糖蛋白,它能增強(qiáng)NK細(xì)胞的活性,對(duì)腎細(xì)胞癌、黑色素瘤、結(jié)腸癌、直腸癌有明顯療效,但過(guò)量使用IL-2會(huì)帶來(lái)不良反應(yīng)。ProTα和IL-2存在緊密的免疫刺激協(xié)同作用,proTα通過(guò)調(diào)節(jié)IL-2誘導(dǎo)的NK敏感細(xì)胞和LAK細(xì)胞毒性,使IL-2的有效濃度降低一半。它們協(xié)同于自體瘤的效果明顯強(qiáng)于單獨(dú)使用IL-2的效果。ProTα和IL-2聯(lián)合使用不但能提高抗腫瘤的效果,還可降低單獨(dú)使用IL-2產(chǎn)生的毒副作用。 本研究利用PCR技術(shù)從前期構(gòu)建好的pcDNA3.1-PTI重組質(zhì)粒中克隆出proTα-linker-IL-2融合基因,并對(duì)其進(jìn)行改造,在5′端加入粒細(xì)胞-巨噬細(xì)胞集落刺激因子(GM-CSF)的信號(hào)肽序列。將改造好的proTα-linker-IL-2融合基因(PI)克隆至真核載體pVAX中構(gòu)建成重組質(zhì)粒pVAX-PI,再將重組質(zhì)粒轉(zhuǎn)化大腸桿菌DH5α,通過(guò)菌落PCR和酶切篩選陽(yáng)性單菌落,提取質(zhì)粒進(jìn)行測(cè)序鑒定。測(cè)序結(jié)果顯示,改造后的融合基因序列與預(yù)期序列一致。 提取無(wú)內(nèi)毒素的重組質(zhì)粒pVAX-PI并定量分析。采用脂質(zhì)體轉(zhuǎn)染試劑將重組質(zhì)粒轉(zhuǎn)染哺乳動(dòng)物細(xì)胞COS-7中進(jìn)行表達(dá),分別收集質(zhì)粒轉(zhuǎn)染細(xì)胞后24h、48h、72h、96h的細(xì)胞上清,用ELISA法定量分析細(xì)胞上清中IL-2蛋白表達(dá)量,結(jié)果顯示,24h、48h、72h、96h轉(zhuǎn)染上清中的IL-2蛋白含量分別為5.4、7.8、6.1、5.7ug/L。采用CTLL-2依賴(lài)細(xì)胞株測(cè)得轉(zhuǎn)染后24h、48h、72h、96h細(xì)胞上清中的IL-2活性分別為43、58、54、49IU/ml。將小鼠原代脾細(xì)胞與細(xì)胞轉(zhuǎn)染上清一起培養(yǎng)48h后,MTT實(shí)驗(yàn)結(jié)果表明,轉(zhuǎn)染上清能刺激小鼠脾細(xì)胞增殖,表達(dá)的融合蛋白具有proTα的活性。最后,通過(guò)ECL western blotting分析和鑒定了表達(dá)的融合蛋白的分子量約為31kDa。 本次研究初步發(fā)現(xiàn)pVAX-PI對(duì)小鼠黑色素瘤有一定的抑制作用,并且對(duì)荷瘤鼠體內(nèi)細(xì)胞因子IFN-γ和IL-4均有一定的誘生作用。pVAX-PI質(zhì)粒免疫組的小鼠外周血清中IFN-γ和IL-4的含量隨時(shí)間的推移而逐漸上升。荷瘤小鼠接種pVAX-PI質(zhì)粒后第5周,ELISA定量分析小鼠外周血清中IFN-γ和IL-4的含量,肌肉注射聯(lián)合在體電脈沖刺激組分別為222pg/ml和157pg/ml,單純肌肉注射組分別為129pg/ml和68pg/ml。以上結(jié)果說(shuō)明,pVAX-PI質(zhì)粒在荷瘤鼠的免疫體系中具有一定調(diào)節(jié)作用。
[Abstract]:Human thymosin 偽 proosin 偽 (Thymosin 偽 prot 偽) is an acidic protein that promotes cell proliferation and has significant immunostimulatory activity. It is mainly used in the treatment of tumor and hepatitis. Interleukin 2 interleukin 2 (IL 2) is a glycoprotein that enhances the activity of NK cells in renal cell carcinoma, melanoma, and colon cancer. Rectal cancer has obvious curative effect, but excessive use of IL-2 can bring adverse reaction. ProT 偽 and IL-2 have close immune stimulation synergism. ProT 偽 regulates the cytotoxicity of NK sensitive cells and LAK cells induced by IL-2. The effective concentration of IL-2 was reduced by half. They were more effective than those of IL-2 alone. Prot 偽 and IL-2 could not only improve the anti-tumor effect. It also reduces the side effects of IL-2 alone. In this study, proT 偽 -linker-IL-2 fusion gene was cloned from pcDNA3.1-PTI recombinant plasmid constructed by PCR technique and modified. The signal peptide sequence of granulocyte-macrophage colony stimulating factor (GM-CSF) was added to the 5'terminal. The modified proT 偽 -linker-IL-2 fusion gene was modified. The recombinant plasmid pVAX-PI was constructed by cloning into eukaryotic vector pVAX. The recombinant plasmid was transformed into Escherichia coli DH5 偽. The positive single colony was screened by colony PCR and enzyme digestion. The plasmid was sequenced and identified. The modified fusion gene sequence was consistent with the expected sequence. The recombinant plasmid pVAX-PI without endotoxin was extracted and quantitatively analyzed. The recombinant plasmid was transfected into mammalian COS-7 by liposome transfection reagent for expression. The supernatants of the cells were collected at 24 h, 48 h, 72 h and 96 h after transfection, and the expression of IL-2 protein in the supernatant was quantitatively analyzed by ELISA method. The results showed that the expression of IL-2 protein was 24 h after transfection. The content of IL-2 protein in the supernatant of 48h / 72h / 96h was 5.4g / L respectively. 24 hours after transfection with CTLL-2 dependent cell line, the content of IL-2 protein was 5.7U / L, respectively. The activity of IL-2 in the supernatant of mouse primary spleen cells and the supernatant of murine primary spleen cells was cultured for 48 h. The results of MTT assay showed that the transfected supernatant could stimulate the proliferation of mouse spleen cells, and the expressed fusion protein had the activity of proT 偽. The molecular weight of the expressed fusion protein was about 31 kDa by ECL western blotting. In this study, we found that pVAX-PI has a certain inhibitory effect on melanoma in mice. Moreover, the levels of IFN- 緯 and IL-4 in peripheral serum of mice immunized with pVAX-PI plasmid were induced by IFN- 緯 and IL-4 in tumor-bearing mice over time. The tumor-bearing mice were inoculated with pVAX-PI plasmid for 5 weeks. The levels of IFN- 緯 and IL-4 in peripheral serum of mice were determined by ELISA. The results of intramuscular injection combined with electrical pulse stimulation in vivo were 222 PG / ml and 157 PG / ml, respectively. The results showed that pVAX-PI plasmid could regulate the immune system of tumor-bearing mice.
【學(xué)位授予單位】：廣西師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R392;Q78

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 易悅;重組大熊貓白介素2和6及其融合蛋白在小鼠模型中對(duì)犬瘟熱重組疫苗的佐劑效應(yīng)研究[D];四川農(nóng)業(yè)大學(xué);2012年

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本文編號(hào)：1437931