本文選題：VP3 + HN　； 參考：《吉林大學》2007年碩士論文

【摘要】： 本研究利用分子生物學、分子免疫學、分子病毒學、生物化學等技術(shù)和手段進行了抗腫瘤治療性重組核酸疫苗的構(gòu)建及純化工藝研究。 首先將三個抑瘤機制不同的抗腫瘤基因,即雞貧血病毒VP3基因、新城疫病毒HN基因及IL-18基因,克隆到真核表達載體pVAX1中,構(gòu)建含有三種基因的真核重組表達質(zhì)粒pVVP3IL-18HN。將重組表達質(zhì)粒pVVP3IL-18HN轉(zhuǎn)染人肝癌SMMC7221細胞,通過RT-PCR、western-blot及間接免疫熒光進行分析,結(jié)果表明,外源基因VP3基因及IL-18HN嵌合基因在SMMC7221細胞中獲得了正確的表達。 將已構(gòu)建的抗腫瘤重組質(zhì)粒(pVVP3IL-18HN),用發(fā)酵罐優(yōu)化發(fā)酵工藝,通過陰離子交換層析方法(Q Sepharose XL)進行純化,將得到的質(zhì)粒DNA通過Source 15Q進行進一步的純化及脫鹽,濃縮。質(zhì)量鑒定結(jié)果表明,通過本研究建立的制備工藝純化獲得的質(zhì)粒DNA,外觀澄清透明,無絮狀顆粒;pH值為7.2±0.2;超螺旋質(zhì)粒比例為87.5%;轉(zhuǎn)化效率為6.20×10~5(cfu/μg質(zhì)粒);純度(OD 260/280)為1.86;未檢測到宿主蛋白質(zhì)、宿主基因組、RNA。本實驗證明了所建立制備工藝的可行,為DNA疫苗的大規(guī)模生產(chǎn)提供了可借鑒的技術(shù)數(shù)據(jù)。
[Abstract]:In this study, molecular biology, molecular immunology, molecular virology, biochemistry and other techniques were used to construct and purify the recombinant nucleic acid vaccine of anti tumor therapeutic recombinant nucleic acid.
First, three antitumor genes, chicken anemia virus VP3 gene, Newcastle disease virus HN gene and IL-18 gene, were cloned into eukaryotic expression vector pVAX1, and the recombinant expression plasmid pVVP3IL-18HN. containing three genes was constructed to transfect the recombinant expression plasmid pVVP3IL-18HN into human hepatoma SMMC7221 cells by RT-PCR, weste Rn-blot and indirect immunofluorescence analysis showed that the foreign gene VP3 and IL-18HN chimeric genes were correctly expressed in SMMC7221 cells.
The constructed anti tumor recombinant plasmid (pVVP3IL-18HN) was optimized by the fermentation tank and purified by the anion exchange chromatography (Q Sepharose XL). The obtained plasmid DNA was further purified and desalted and concentrated by Source 15Q. The quality identification results showed that the preparation process was obtained through the preparation process of this study. Plasmid DNA was clear and transparent with no floc particles, pH value was 7.2 + 0.2, the ratio of super helix plasmid was 87.5%, the conversion efficiency was 6.20 x 10~5 (cfu/ g plasmid), and the purity (OD 260/280) was 1.86. The host protein and host genome were not detected, and RNA. experiment proved that the preparation process was feasible, which provided the large scale production of DNA vaccine. Technical data for reference.

【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392

【參考文獻】

相關(guān)期刊論文 前10條

1 張志珍,毛積芳,楊生生,竇鴻;高密度培養(yǎng)大腸桿菌表達重組人胰高血糖素樣肽-1[J];第二軍醫(yī)大學學報;2002年03期

2 林懿,陳祖歡,郭瀛軍,孫樹漢;高密度培養(yǎng)大腸桿菌生產(chǎn)重組鉤端螺旋體DNA疫苗[J];第二軍醫(yī)大學學報;2002年08期

3 雷涵,鄧樂,朱文杰;亞微米生物功能磁性復合微球的構(gòu)建及在質(zhì)粒DNA捕獲中的應用[J];高技術(shù)通訊;2002年08期

4 朱林;白細胞介素18與腫瘤[J];國外醫(yī)學(免疫學分冊);2004年03期

5 柯偉;夏杰;陸兵;徐殿勝;;重組大腸桿菌發(fā)酵生產(chǎn)核酸疫苗的初步研究[J];華東理工大學學報(自然科學版);2006年01期

6 劉文華,肖祥華,施產(chǎn)甫,徐根興,傅更鋒,樊燕蓉,劉新卷;pcDNA3-內(nèi)皮抑素質(zhì)粒的大規(guī)模純化[J];南京軍醫(yī)學院學報;2003年03期

7 王志軍,樂國偉,施用暉,汪垣;營養(yǎng)條件對pcDNA3-HBS質(zhì)粒DNA生產(chǎn)的影響[J];生物工程學報;2001年03期

8 孫敏莉,張皓;納米磁性粒子在DNA分離與純化中的應用進展[J];生物工程學報;2001年06期

9 程海;唐欣昀;劉勇;;臨床用DNA疫苗生產(chǎn)工藝研究進展[J];微生物學免疫學進展;2006年02期

10 周郁,周仲毅;腫瘤細胞因子基因治療的研究現(xiàn)狀[J];西北國防醫(yī)學雜志;2001年01期

，

本文編號：1859389