【摘要】： 目的提高16型人乳頭瘤病毒(HPV16)L1基因以桿狀病毒為載體,在昆蟲細(xì)胞中的表達(dá)水平,為研制預(yù)防性HPV疫苗奠定基礎(chǔ)。 方法根據(jù)昆蟲細(xì)胞密碼子偏性對野生型HPV16L1基因進(jìn)行改造,全基因合成密碼子優(yōu)化型HPV16L1基因,利用Bac-to-Bac表達(dá)系統(tǒng)獲得重組桿狀病毒,感染昆蟲細(xì)胞Sf9和High Five。Western blot鑒定表達(dá)產(chǎn)物；電鏡下觀察病毒樣顆粒形成。利用ELISA法評價(jià)HPV16L1基因的優(yōu)化效果,并探討L1蛋白在兩種昆蟲細(xì)胞中表達(dá)的最佳條件。 結(jié)果Sf9和High Five細(xì)胞的表達(dá)產(chǎn)物經(jīng)Western blot檢測,在相對分子質(zhì)量56 kD處均出現(xiàn)HPV16L1的特異性條帶；電鏡下可見病毒樣顆粒在兩種昆蟲細(xì)胞的核內(nèi)形成；ELISA法結(jié)果顯示密碼子優(yōu)化型HPV16L1基因的表達(dá)水平顯著高于野生型(P0.01)。High Five細(xì)胞表達(dá)的最佳條件為MOI=10,表達(dá)時(shí)相72 h,其L1蛋白表達(dá)量至少比Sf9細(xì)胞高3倍。 結(jié)論密碼子優(yōu)化技術(shù)確實(shí)能夠促進(jìn)HPV16L1蛋白的高效表達(dá),而High Five細(xì)胞表現(xiàn)出的顯著優(yōu)勢尤其值得關(guān)注。
[Abstract]:Objective to improve the expression level of L1 gene of human papillomavirus type 16 (HPV16) in insect cells using baculovirus as vector, so as to lay a foundation for the development of prophylactic HPV vaccine. Methods based on the codon bias of insect cells, the wild type HPV16L1 gene was modified, the codon optimized HPV16L1 gene was synthesized, and the recombinant baculovirus was obtained by using the Bac-to-Bac expression system. The expression products were identified by Sf9 and High Five.Western blot in infected insect cells. Virus-like particles were observed under electron microscope. ELISA method was used to evaluate the optimal effect of HPV16L1 gene expression and the optimal conditions for L1 protein expression in two insect cells were discussed. Results the expression products of Sf9 and High Five cells were detected by Western blot, and the specific bands of HPV16L1 were found at the relative molecular weight of 56 kD, and the virus like particles were formed in the nuclei of the two insect cells under electron microscope. The results of ELISA showed that the expression level of codon optimized HPV16L1 gene was significantly higher than that of wild type (P0.01). High Five cells expressed at 72 h after MOI=10, expression, and the expression of L1 protein was at least 3 times higher than that of Sf9 cells. Conclusion the codon optimization technique can promote the high expression of HPV16L1 protein, and the significant advantage of High Five cells is worthy of attention.
【學(xué)位授予單位】：沈陽藥科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392.1;Q78

【參考文獻(xiàn)】

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1 任占平,黃健輝,石U，

本文編號：2321157