新型慢病毒包装方法及转基因鸡生物反应器研究

发布时间：2018-07-10 16:30

本文选题：动物生物反应器 + 慢病毒　；参考：《南京农业大学》2016年硕士论文

【摘要】：近十几年来,转基因鸡的制备在人们的不懈努力下,取得了长足的进步。这一领域之所以能被人们广为关注,不仅仅因为鸡是一种理想的模式动物,更为重要的是转基因鸡可以改造成为高效生产人源药用蛋白的生物反应器。禽输卵管生物反应器比传统工业反应器和哺乳动物细胞生物反应器更加高效而廉价已经成为业界的共识。这也使转基因鸡的研究成为科研领域的一大热点和生物制药行业发展的新方向。由于鸡胚胎发育的特殊性,在转基因鸡生物反应器的制备中必需使用有别于哺乳类动物生物反应器的特殊手段。目前看来,使用慢病毒载体制备转基因鸡是最可靠的实验方法。但是由于传统慢病毒包装方法的巨大缺陷,以及鸡胚注射和后续筛选中存在的诸多问题,使得成功制备转基因鸡生物反应器的案例仍然较少被报道。在本实验的第一部分中,我们使用了以聚乙烯亚胺(PEI)为转染介质,以聚乙二醇6000(PEG6000)为离心浓缩介质的慢病毒生产方法,并对浓缩后的慢病毒进行滴度测定。此外也对可能影响慢病毒滴度的几个关键因素如转染初始细胞数、N/P、转染质粒DNA量、佐剂的选择等的作用进行了分析。实验结果说明在使用15cm培养皿生产慢病毒的条件下,转染质粒DNA总量10.5μg,N/P=20,PEI浓储液pH7.0-9.0,转染时293T细胞数0.5-2×107时,可以获得较好的慢病毒滴度。PEI作为一种新的转染试剂,可以替代磷酸钙和脂质体用于高滴度慢病毒的生产。在第二和第三部分中,我们通过对现有鸡胚开窗注射方法诸多细节的不断优化,以及后续的一系列检测手段,最终成功获得生殖腺嵌合型转基因鸡。实验结果说明,通过封口方式、封口膜种类、胚胎穿刺位置、开窗操作时间、开窗后的液封处理、防止污染的措施等方面的改进,我们将开窗注射孵化率提高到了 55%。在出雏个体中,转基因嵌合型个体所占比例提高到了 41.8%。在对所获得的31只嵌合体鸡进一步筛选后,我们最终获得了 4公4母共8只生殖腺嵌合型转基因鸡。综上所述,本实验研究表明,PEI作为一种新的转染试剂,可以替代磷酸钙和脂质体用于高滴度慢病毒的生产。与二者相比,PEI具有更低的操作门槛和更高的性价比,从而具有很高的使用价值,在未来一定具有广阔的使用前景。同时本实验也详细介绍了一套完整的制备转基因鸡输卵管生物反应器的方法,希望对广大科研人员有所帮助。
[Abstract]:In the past ten years, the preparation of transgenic chicken has made great progress under the unremitting efforts of people. The reason why this field can be widely concerned is not only because chicken is an ideal model animal, but also because transgenic chicken can be transformed into a bioreactor that can efficiently produce human medicinal protein. Avian fallopian tube bioreactor is more efficient and cheaper than traditional industrial reactor and mammalian cell bioreactor. This also makes the research of transgenic chicken become a hot spot in the field of scientific research and a new direction of the development of biopharmaceutical industry. Because of the particularity of chicken embryo development, it is necessary to use special methods different from mammalian bioreactor in the preparation of transgenic chicken bioreactor. At present, using lentivirus vector to prepare transgenic chicken is the most reliable experimental method. However, due to the huge defects of the traditional lentivirus packaging method and the problems in chicken embryo injection and subsequent screening, the successful preparation of transgenic chicken bioreactor is still rarely reported. In the first part of this experiment, we used polyvinyleneimine (PEI) as transfection medium and polyethylene glycol 6000 (PEG6000) as centrifugal concentration medium to produce lentivirus, and determined the titer of concentrated lentivirus. In addition, the effects of several key factors which may affect the titer of lentivirus such as the number of transfection cells N / P, the amount of plasmid DNA transfection, the selection of adjuvant were analyzed. The results showed that under the condition of producing lentivirus by using 15cm culture dish, a better lentivirus titer. PEI could be obtained as a new transfection reagent under the condition that the total amount of the transfected plasmid was 10.5 渭 g / N ~ (-1) N ~ (-1) N ~ (-1) N ~ (20) P ~ (20) P ~ (-1) and 293T cell number was 0.5-2 脳 10 ~ 7, respectively. Calcium phosphate and liposomes can be substituted for the production of high titer lentivirus. In the second and third parts, we successfully obtained the genitonal chimeric transgenic chicken by optimizing the details of the existing chicken embryo fenestration injection method and a series of subsequent detection methods. The experimental results show that through the improvement of the sealing mode, the type of sealing membrane, the location of embryo puncture, the operating time of window opening, the treatment of liquid seal after window opening, and the measures to prevent pollution, we have raised the hatching rate of window opening injection to 5555%. The proportion of transgenic chimeric individuals increased to 41.8%. After further screening of 31 chimeric chickens, we finally obtained 4 males, 4 females and 8 gonadal chimeric transgenic chickens. In conclusion, as a new transfection reagent, PEI can replace calcium phosphate and liposome in the production of high titer lentivirus. Compared with the two methods, PEI has a lower operating threshold and a higher performance-to-price ratio, so it has a high value of use, and will certainly have a broad application prospect in the future. At the same time, a complete method of preparing transgenic chicken fallopian tube bioreactor was introduced in detail.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q81

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