幾丁聚醣接枝犬尿酸/DNA奈米微粒在基因轉殖雞上的應用

Abstract

現在有許多蛋白質藥物量小且價格昂貴，生產的方式不外乎將所欲表現蛋白質的外源基因利用各種方式嵌入微生物（如E.coli、酵母菌等），但不能夠大量生產，且還有許多外源基因在微生物上的表現會受限、耗時費力等問題存在，所以，在基因轉殖動物的研究便成為人們注意的新焦點。 目前在基因轉殖雞的研究上，發現雞不但可以克服哺乳動物上會產生的缺點，而且還有許多額外的優勢在。 本研究在幾丁聚醣上接犬尿酸來當作一新的基因載體，並做出粒徑150~300 nm穩定均勻的奈米微粒，且DNA包覆率可達90 %以上。在N/P=2時，界面電位可達+9 mV，且比未改質幾丁聚醣更能有效保護DNA免於限制脢的切割。改質過後的幾丁聚醣對細胞並無負面影響，且能在3T3及HeLa細胞有轉染的表現，但3T3細胞轉染效果明顯優於HeLa，顯示基因載體對不同細胞的轉染能力不同。最後在雞胚於第0日時將此改質過後的基因載體注入雞胚，發現雞隻於奈米微粒注入後存活率剩下14%，且沒有綠色螢光表現，但改質後幾丁聚醣/DNA奈米微粒仍具有許多優勢可作為基因轉殖雞研究的載體。

There are many therapeutic proteins and peptide have been licensed for production using bacterial, fungal and mammalian cells, and many therapeutic proteins are currently being developed and tested in a variety of host cells. Transgenic animals describes animals with chromosomes that contain stably integrated copies of genes. The use of transgenics as a technology for producing recombinant proteins has made remarkable strides in the past few years. Recently, transgenic chickens should be near-perfect bioreactors for making large amounts of pure recombinant proteins. In this study, we tried to use urocanic acid to modify chitosan as a new vector in gene delivery. We made the nanoparticles with diameter is 150~300 nm, and the DNA loading efficiency is over 90 %. When N/P=2, the zeta potential is +9 mV. And the modified chitosan/DNA nanoparticles can protect the DNA efficiently than unmodified chitosan/DNA. The modified chitosan had very good biocompatibility in 3T3 and HeLa, but the transfection efficiency is cell-dependant. In vivo test, the viability of the treated chicken embryos was down to 14%, and no any green fluroence protein was observed. But the modified chitosan/DNA nanoparticle still has a lot of advantages which applied in transgenic chicken.