基因轉殖斑馬魚作為生物反應器表現人類凝血蛋白

Zebrafish as bioreactors to produce human coagulation factors

黃瀚毅(Han-I Huang)

指導教授 ： 蔡懷楨

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.10966

摘要

A型血友病(Hemophilia A)是一種因血液凝固機制發生異常的遺傳性出血性疾病，主要是因為人類第八凝血因子(human factor VIII, hFVIII)的缺乏所導致，目前醫療技術無法完全根治，必須長期接受治療。現今研究是以基因轉殖工程技術讓生物反應器(bioreactors)製備非血液純化的hFVIII。主要為利用動物細胞株，例如倉鼠卵巢細胞株(Chinese hamster ovary cells, CHO cells)或倉鼠腎細胞株(Baby hamster kidney cells, BHK cells)製備重組hFVIII，然而培養槽容納細胞數量有限，提升規模所需費用昂貴導致成本過高。若選用基因轉殖動物，例如豬或牛來製備重組hFVIII，飼育時間則過於冗長，也相對提高成本。而斑馬魚生命週期較短、產卵數多，在控溫情況下終年可產卵，加上基因轉殖技術容易，養殖所需設備簡單成本便宜。因此本研究欲使用斑馬魚當作bioreactor，於斑馬魚體內及魚卵中製備重組hFVIII。本研究首先構築分別由zebrafish hsp 70/4 promoter或zebrafish a-tubulin promoter或CMV promoter來驅動移除了釋泌訊息胜肽(signal peptide)與hFVIII B domain的hBDD-FVIII基因片段或hBDD-FVIII-EGFP基因片段。接著將CMV promoter驅動hBDD-FVIII基因片段的表現載體透過細胞轉染到HEK293T細胞株中，用Western blot偵測表現的細胞株蛋白質，可偵測到接近170 KDa大小左右的hBDD-FVIII重組蛋白質。發現移除了signal peptide與B domain的hBDD-FVIII仍可以在哺乳類動物細胞株內表現。然後藉由顯微注射將zebrafish hsp 70/4 promoter與zebrafish a-tubulin promoter驅動hBDD-FVIII基因片段或hBDD-FVIII-EGFP基因片段的表現載體轉殖到斑馬魚胚胎中，並利用綠螢光表現有無篩選可能基因轉殖品系G0親代。其中由zebrafish hsp 70/4 promoter驅動hBDD-FVIII基因片段的基因轉殖斑馬魚G0親代已達性成熟，育成後與野生型斑馬魚配對，挑選子代有綠螢光表現的基因轉殖品系G0親代，並利用PCR檢測基因轉殖品系其F1子代胚胎的genomic DNA，可以偵測到約為預期547 bp大小的hBDD-FVIII外源基因片段訊號。目前篩選出9隻基因轉殖斑馬魚G0其子代有綠螢光表現並且genomic DNA中確認帶有hBDD-FVIII外源基因片段，將這9隻基因轉殖斑馬魚G0與野生型斑馬魚配對並收集有綠螢光表現的F1子代胚胎進行飼育。之後進一步收集25隻基因轉殖品系其F1子代胚胎進行熱誘導處理，透過RT-PCR檢測熱誘導處理後基因轉殖品系G0其F1子代胚胎的mRNA，同樣可以偵測到如預期547 bp大小的hBDD-FVIII外源基因片段訊號。本研究確認基因轉殖斑馬魚品系其F1子代胚胎經由熱誘導處理後，hBDD-FVIII外源基因片段不僅在genomic DNA中順利遺傳，在RNA層次上hBDD-FVIII外源基因片段也能藉由熱誘導處理後順利轉錄成hBDD-FVIII mRNA。然而利用Western blot偵測熱誘導處理之基因轉殖品系其F1子代胚胎全蛋白，以人類FVIII heavy chain抗體並未偵測到訊號，顯示在蛋白質層次上，hBDD-FVIII外源基因片段於熱誘導處理後並未能順利轉譯出hBDD-FVIII重組蛋白質。

關鍵字

斑馬魚 ； 生物反應器 ； 凝血蛋白

並列摘要

Hemophilia A is an X-linked, recessive bleeding disorder, which is caused by the defective human coagulation factor VIII (hFVIII). At the present time, Hemophilia A patients are treated with recombinant hFVIII produced by chinese hamster ovary (CHO) cells or baby hamster kidney (BHK) cells. However, it is very costly. In this study, we attempted to use zebrafish (Danio rerio) as a bioreactor to produce recombinant hFVIII because of short generation time, high fecundity, simple and cheap culture system, and easy manipulation of gene transfer. We constructed an expression plasmid, in which B domain-deleted hFVIII (hBDD-FVIII) was driven by either CMV promoter. After HEK293T cells were transfected with pCMV-BDFVIII, the total extracted proteins were subjected to western blot analysis using antiserum against hFVIII heavy chain. Results showed that there was a positive band located at 170 kDa, which was corresponding to the recombinant hBDD-FVIII protein. Other expression plasmids pHBDFVIII, pHBDFVIII-EGFP and pZaBDFVIII-EGFP were also constructed, in which either hBDD-FVIII or hBDD-FVIII-EGFP was driven by zebrafish hsp 70/4 promoter or zebrafish a-tubulin promoter, and microinjected into one-celled zebrafish embryos individually. In total, nine G0 lines harboring pHBDFVIII were generated. We extracted genomic DNA from F1 embryos and detected by PCR. A 547 bp PCR-product was amplified and corresponded with the amplification of partial hFVIII A3 domain from transgene. We also extracted total RNA from F1 embryos treated by heat-shock treatment and detected by RT-PCR. A 547 bp PCR-product was also amplified and corresponded with the amplification of transgene, suggesting that these lines can transcribe the recombinant B domain-deleted human factor VIII transgene after induction. Furthermore, the total proteins that were extracted from F1 embryos treated by heat-shock treatment were subjected to western blot analysis using antiserum against hFVIII heavy chain. However, results showed that there was no signal corresponding to the recombinant hBDD-FVIII protein. It suggested that these lines can not translate the recombinant B domain-deleted human factor VIII protein after induction.

並列關鍵字

zebrafish ； bioreactor ； coagulation factor

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