於昆蟲細胞中運用雙效表現載體以桿狀病毒轉錄活化因子或人類XBP-1S來增益重組蛋白質之產量

Evaluate baculovirus encoded transcriptional factors or hXBP-1S that can enhance recombinant protein production in insect cells through a novel bi-cistronic expression vector

廖騏宏(Chi-Hung Liao)

指導教授 ： 吳宗遠

中原大學/工學院/生物科技研究所/碩士(2009年)

https://doi.org/10.6840/cycu200900966

摘要

本研究中，將試驗四個不同的轉錄活化因子：分別為桿狀病毒極早期蛋白質（IE1、IE2 以及PE38），以及人類X‐box‐binding‐protein 1 （hXBP‐1），並在昆蟲細胞中，利用雙效表現載體表現這些轉錄活化因子，評估哪一轉錄活化因子對於增益以internal ribosome entry site（IRES） 調節之重組蛋白的產量是最具潛力的。在感染桿狀病毒的Sf21細胞中，IE1、IE2以及PE38蛋白質可以trans‐activate多種不同時期桿狀病毒基因表現；而hXBP‐1S在內質網緊迫（ER stress）之下，是為轉錄活化因子，可以調節、活化許多種內質網內的監護子（chaperones）。當我們將欲生產之重組蛋白質分別與上述四種轉錄活化因子同時表現時，推測可以增加目標基因之轉錄或是增益此重組蛋白質之品質，藉此增加欲生產之重組蛋白質的產量。首先，構築vAc-X-Rhir-SEFP（X代表這四個不同的轉錄活化因子；SEFP是secretory alkaline phosphatase (SEAP) 及 EGFP 的融合蛋白，作為一分泌性的報導基因，並以RhPV IRES（來自Rhopalosiphum padi virus）調控其表現。重組病毒感染Sf21細胞株， 證實hXBP-1S蛋白質可以提升SEFP報導蛋白質的表現量，而其他桿狀病毒的轉錄活化因子提升效果未及hXBP-1S。因此，利用雙效表現載體以啟動子與RhPV IRES同時表達hXBP-1S蛋白質，是可以有效地增益此重組蛋白質之產量。接著在非裂解性昆蟲細胞表現系統中，測試這些轉錄活化子的功效，構築pIB-X-Pnir-SEFP （以Orgyia pseudotsugata multiple nucleopolyhedro virus （OpMNPV）的ie2啟動子表達轉錄活化因子；Pnir是代表PnV IRES （來自 Perina nuda Iflavirus））並與另一質體pIB-lac Z同時轉移入Sf9細胞株中，藉此測試這些轉錄活化子。我們發現在在非裂解性昆蟲細胞表現系統中，只有IE2才能有效的增加重組蛋白質的表現量。這個桿狀病毒IE2能cis-activate SEAP分泌性蛋白質活性至2到3倍，而trans-activate β- galatosidase活性至六倍左右，其它的轉錄活化因子則沒有顯著地功效。總之hXBP‐1S在桿狀病毒表現系統中能有效地增益重組蛋白質的產量；而桿狀病毒IE2蛋白質可以在非裂解性昆蟲細胞表現系統中，有效地增益重組蛋白質的產量。

關鍵字

轉錄因子 ； 分泌性綠螢光融合蛋白 ； 核醣體內轉譯子 ； 雙效表現載體 ； 昆蟲細胞表現系統

並列摘要

In this study, we tested four transcription activators, three baculoviruses immediate early proteins (IE1, IE2 and PE38) and a human X‐box‐binding‐protein 1 (hXBP‐1), to evaluate which can increase the production of recombinant protein through a bi-cistronic vectors. IE1, IE2 and PE38 proteins can trans‐activate several baculovirus genes expression in baculovirus infected cells and the human hXBP‐1S has been shown that can act as an transcriptional activator during ER stress and can also activate the expression of many ER chaperones. We hypothesized that when the targete protein was co-expressed with IE1, IE2, PE38 or XBP‐1S could enhance the targeting gene transcription or increase the protein quality and resulted in the high production of the interest recombinant protein. First of all, we employed BEVS to test these transcriptional factors efficiencies in baculovirus infected Sf21 cells. We constructed the recombinant virus vAc-X-Rhir-SEFP（X means the four transcriptional factors that were expressed by polyhedrin promoter, and SEFP（a fusion protein of secretory alkaline phosphatase (SEAP) and EGFP ）as a secretory reporter gene following the RhPV IRES（from Rhopalosiphum padi virus）to infect with Sf21 cells. Our results demonstrated that the hXBP-1S protein rather than other transcriptional factors can enhance the expression of sefp gene. These results indicated that bi-cistronic expression vector containing the RhPV IRES can simultaneously express hXBP-1S proteins with gene of interest (GOI) and enhance the recombinant proteins production in the baculovirus expression vector system. Second, we used the non-lytic insect cell expression system to test these transcriptional factors to find the enhancing ability on recombinant protein production in the absence of baculovirus infection. The plasmid pIB-X-Pnir-SEFP drived by the ie2 promoter of Orgyia pseudotsugata multiple nucleopolyhedro- virus (OpMNPV), and Pnir ( PnV IRES derived from Perina nuda Iflsvirus）were co-transfected with this plasmid pIB-lac Z into Sf9 cells. This result showed that only the IE2 protein can enhance the recombinant protein production. The baculovirus IE2 protein can cis-activate SEFP activity up to two to three folds, and it can trans-activate β-gal activity up to six folds than other transcriptional factors. In conclusion, our results indicate the hXBP‐1S can up-regulate the recombinant protein production in the baculovirus expression vector system and IE2 was optimal in the non-lytic insect cell expression system.

並列關鍵字

transcriptional factors. ； internal ribosome entrysite (IRES) ； insect cell expression system ； secretory alkaline phosphatase (SEAP) and EGFP fusion p ； bi-citronic expression vector

參考文獻

林以婷 (2008). 發展非裂解性昆蟲細胞雙效表現載體. 私立中原大學碩士學位論文, 桃園.

Ailor, E., and Betenbaugh, M.J. (1999). Modifying secretion and post-translational processing in insect cells. In Current opinion in biotechnology, pp. 142-145.

Ayres, M.D., Howard, S.C., Kuzio, J., Lopez-Ferber, M., and Possee, R.D. (1994). The complete DNA sequence of Autographa californica nuclear polyhedrosis virus. Virology 202, 586-605.

Bassaneze, V., Miyakawa, A.A., and Krieger, J.E. (2008). A quantitative chemiluminescent method for studying replicative and stress-induced premature senescence in cell cultures. Anal Biochem 372, 198-203.

Blissard, G.W. (1996). Baculovirus--insect cell interactions. Cytotechnology 20, 73-93.