应用慢病毒介导生产转基因鸡的初步研究

发布时间：2018-06-24 09:46

本文选题：慢病毒 + 胚胎注射　；参考：《广西大学》2017年硕士论文

【摘要】：转基因鸡在畜牧行业不仅是一种重要的经济动物,而且鸡的输卵管生物反应器是生产药物蛋白的理想生物反应器,转基因鸡的生产具有广阔的应用前景。本研究构建了不同的慢病毒载体,并对慢病毒转基因鸡研究的方法进行了系统的探索,取得如下结果:慢病毒转基因载体的构建:在本研究中构建了过表达的GV358-LV-HL F、pLVX-IRES-LTF-EGFP 和 pCDH-CMV-LTF-mcherry 载体慢病毒载体,三个载体以CMV启动子调控表达HLF和LTF的基因序列,通过本实验室的三质粒包装慢病毒系统分别包装出了高滴度的慢病毒,通过倍比稀释的方法检测出慢病毒滴度达到109TU/mL。不同因素对慢病毒转基因制备效率的探索:本研究探索了不同因素对种蛋孵化率的影响。通过不同封口方式试验结果显示:种蛋石蜡封口,出壳率70%。种蛋封口膜封口,出壳率60%。均低于对照组出壳率100%。不同开口位置对鸡胚孵化率的影响。种蛋钝端开口,孵化率为70%。种蛋赤道面开口,孵化率为50%,均低于对照组孵化率为100%。不同口径的注射器械对鸡胚孵化率的影响。1mL注射器注射种蛋,孵化率为60%。20μL微量注射器注射种蛋,孵化率为60%。40um 口径的显微注射针注射种蛋,孵化率为70%。均低于对照组孵化率100%。赤道面开口注射不同溶液对鸡胚发育的损伤。台盼蓝注射种蛋,孵化率为0%。PBS注射种蛋,孵化率为25%。生理盐水注射种蛋,孵化率为30%。均低于对照组孵化率100%。慢病毒制备转基因鸡:钝端注射不同滴度慢病毒对鸡胚发育的影响。慢病毒滴度108TU/mL注射种蛋,孵化率50%。慢病毒滴度107TU/mL注射种蛋,孵化率50%。慢病毒滴度106TU/mL注射种蛋,孵化率65%。慢病毒滴度105TU/mL注射种蛋,孵化率65%。生理盐水组孵化率70%。赤道面用109TU/mL慢病毒液注射种蛋,出壳数10只,孵化率50%。慢病毒转基因鸡胚胎和鸡的检测:提取血液和组织DNA,PCR检测。钝端注射制备转基因鸡的方法未检测到阳性的转基因鸡。赤道面注射制备转基因鸡的方法在鸡喙、胰腺、脑、肌胃、肺脏、心脏、法氏囊、肾脏检测目的基因的表达。本研究通过赤道面显微注射获得了 G0表达LTF的阳性转基因鸡,为以后转基因鸡的生产提供了参考,为应用慢病毒介导方法生产鸡输卵管生物反应器研究奠定了良好的基础。
[Abstract]:Transgenic chicken is not only an important economic animal in livestock industry, but also an ideal bioreactor for producing pharmaceutical protein in oviduct bioreactor of chicken. In this study, different lentivirus vectors were constructed, and the methods of lentivirus transgenic chicken were systematically explored. The results are as follows: the construction of lentivirus transgenic vector: in this study, the overexpression of GV358-LV-HL FGV pLVX-IRES-LTF-EGFP and pCDH-CMV-LTF-mcherry vector lentivirus vectors were constructed. The three vectors expressed HLF and LTF gene sequences regulated by CMV promoter. The lentivirus with high titer was packaged by the three plasmids packaging lentivirus system in our laboratory. The titer of lentivirus was detected by double dilution method and the titer of lentivirus was 109TU / mL. The effects of different factors on the hatching rate of lentivirus were studied. The results of different sealing methods showed that the shell rate was 70% after paraffin sealing. Seed egg sealing membrane sealing, shell rate 60. All of them were lower than that of the control group (100%). Effect of different opening position on hatching rate of chicken embryo. The hatching rate is 70. The hatching rate was 50%, which was lower than that of the control group (100%). Effect of different caliber injection equipment on hatching rate of chicken embryo. 1 mL injector injected egg, hatching rate was 60.20 渭 L microinjector, and hatching rate was 60%.40um caliber microinjection needle, hatching rate was 70. The hatching rate was 100% lower than that of the control group. Damage to chicken embryo development caused by injection of different solutions into the equatorial opening. The hatching rate of Trypan blue was 0. PBS and the hatching rate was 25. The hatching rate was 30%. The hatching rate was 100% lower than that of the control group. Preparation of transgenic chicken by lentivirus: effects of different titers of lentivirus at blunt end on embryo development. Lentivirus titer 108 tu / mL was injected into the egg, and the hatching rate was 50%. The lentivirus titer 107 tu / mL was injected into the egg, and the hatching rate was 50%. Lentivirus titer 106 tu / mL was injected into the egg, and the hatching rate was 65%. Lentivirus titer 105 tu / mL was injected into the egg, and the hatching rate was 65%. The hatching rate of normal saline group was 70%. The eggs were injected with 109 tu / mL lentivirus solution on the equatorial surface, and the hatching rate was 50. Detection of Lentivirus transgenic Chicken embryos and Chicken: extraction of DNA from Blood and tissue by PCR. Positive transgenic chickens were not detected by blunt end injection. The expression of target gene in beak, pancreas, brain, muscle stomach, lung, heart, bursa of Fabricius and kidney were detected by equatorial injection. In this study, G0 expressing LTF positive transgenic chickens were obtained by equatorial microinjection, which provided a reference for the future production of transgenic chickens, and laid a good foundation for the production of chicken fallopian tube bioreactor by lentivirus-mediated method.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S831

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