本文選題：慢病毒 + 胚胎注射�。� 參考：《廣西大學(xué)》2017年碩士論文

【摘要】：轉(zhuǎn)基因雞在畜牧行業(yè)不僅是一種重要的經(jīng)濟動物,而且雞的輸卵管生物反應(yīng)器是生產(chǎn)藥物蛋白的理想生物反應(yīng)器,轉(zhuǎn)基因雞的生產(chǎn)具有廣闊的應(yīng)用前景。本研究構(gòu)建了不同的慢病毒載體,并對慢病毒轉(zhuǎn)基因雞研究的方法進行了系統(tǒng)的探索,取得如下結(jié)果:慢病毒轉(zhuǎn)基因載體的構(gòu)建:在本研究中構(gòu)建了過表達的GV358-LV-HL F、pLVX-IRES-LTF-EGFP 和 pCDH-CMV-LTF-mcherry 載體慢病毒載體,三個載體以CMV啟動子調(diào)控表達HLF和LTF的基因序列,通過本實驗室的三質(zhì)粒包裝慢病毒系統(tǒng)分別包裝出了高滴度的慢病毒,通過倍比稀釋的方法檢測出慢病毒滴度達到109TU/mL。不同因素對慢病毒轉(zhuǎn)基因制備效率的探索:本研究探索了不同因素對種蛋孵化率的影響。通過不同封口方式試驗結(jié)果顯示:種蛋石蠟封口,出殼率70%。種蛋封口膜封口,出殼率60%。均低于對照組出殼率100%。不同開口位置對雞胚孵化率的影響。種蛋鈍端開口,孵化率為70%。種蛋赤道面開口,孵化率為50%,均低于對照組孵化率為100%。不同口徑的注射器械對雞胚孵化率的影響。1mL注射器注射種蛋,孵化率為60%。20μL微量注射器注射種蛋,孵化率為60%。40um 口徑的顯微注射針注射種蛋,孵化率為70%。均低于對照組孵化率100%。赤道面開口注射不同溶液對雞胚發(fā)育的損傷。臺盼藍注射種蛋,孵化率為0%。PBS注射種蛋,孵化率為25%。生理鹽水注射種蛋,孵化率為30%。均低于對照組孵化率100%。慢病毒制備轉(zhuǎn)基因雞:鈍端注射不同滴度慢病毒對雞胚發(fā)育的影響。慢病毒滴度108TU/mL注射種蛋,孵化率50%。慢病毒滴度107TU/mL注射種蛋,孵化率50%。慢病毒滴度106TU/mL注射種蛋,孵化率65%。慢病毒滴度105TU/mL注射種蛋,孵化率65%。生理鹽水組孵化率70%。赤道面用109TU/mL慢病毒液注射種蛋,出殼數(shù)10只,孵化率50%。慢病毒轉(zhuǎn)基因雞胚胎和雞的檢測:提取血液和組織DNA,PCR檢測。鈍端注射制備轉(zhuǎn)基因雞的方法未檢測到陽性的轉(zhuǎn)基因雞。赤道面注射制備轉(zhuǎn)基因雞的方法在雞喙、胰腺、腦、肌胃、肺臟、心臟、法氏囊、腎臟檢測目的基因的表達。本研究通過赤道面顯微注射獲得了 G0表達LTF的陽性轉(zhuǎn)基因雞,為以后轉(zhuǎn)基因雞的生產(chǎn)提供了參考,為應(yīng)用慢病毒介導(dǎo)方法生產(chǎn)雞輸卵管生物反應(yīng)器研究奠定了良好的基礎(chǔ)。
[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.
【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S831

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