Ndst4基因剔除鼠之表現型分析暨小鼠Ndst4抗血清製備

Abstract

大腸直腸癌生成主要是由於基因變異的累積所造成，如致癌基因的活化和抑癌基因的刪除。本實驗室先前的研究，在人類第四號染色體4q25-4q28.3區域篩選出NDST4於大腸直腸癌可能扮演抑癌基因的角色，為了研究NDST4的功能，實驗室製造Ndst4基因剔除小鼠。本論文主要分為兩個研究，第一部分於Ndst4基因剔除鼠，研究其表現型的變化，以探討此基因於小鼠可能的生理功能。首先，利用多重聚合酶鏈鎖反應(multiplex PCR)和南方墨點法確認Ndst4基因剔除小鼠的基因型，再以反轉錄聚合酶鏈鎖反應確認Ndst4基因在野生型小鼠各器官的表現情況，並利用RNA表現量最高的器官(腦)，確認Ndst4基因剔除鼠之RNA表現。利用全血球計數(complete blood count, CBC)、血清生化檢驗、modified-SHIRPA和組織切片檢查Ndst4基因剔除小鼠是否有表現型的變異。在modified-SHIRPA、CBC和血清生化檢驗的初步結果相較於野生型小鼠，Ndst4基因剔除小鼠並沒有看到顯著的差異，但在組織切片檢查，發現Ndst4基因剔除小鼠在近端大腸之杯狀細胞數量增加，且在小腸絨毛形態改變。論文第二部分是小鼠Ndst4抗血清的製作。首先利用CLC Sequence viewer 6分析軟體找出mNdst4蛋白質與mNdst家族其他成員(Ndst1-Ndst3)相似性最低的一段胺基酸序列，再將此段序列做B細胞抗原決定位(B cell epitope)的分析，最後決定以mNdst4第40-226個胺基酸序列做為抗原(mNdst4-N)，利用E coli.表現目標蛋白並純化。再將純化mNdst4-N蛋白打入五隻Balb/C小鼠，最後以西方墨點法檢測小鼠血清中，anti-mNdst4-N抗體的效價與專一性。結果得知：五隻小鼠之抗血清皆能辨認mNdst4抗原且一號和三號小鼠能與人類NDST4 (hNDST4)產生交叉反應。從目前結果預期：未來能應用於mNdst4和hNDST4專一性之單株抗體的製作，並幫助爾後此蛋白生理功能之研究。

The development of colorectal cancer originates from accumulation of many genetic defects, including activation of oncogenes and deletion of tumor suppressor genes. Our previous study found a putative tumor suppressor gene, NDST4, at chromosome 4q26 in colorectal cancer. So, we produced a Ndst4 knock out (Ndst4 KO) mouse to study the probable physiological functions of NDST4. In this thesis, there are two parts of investigation. First, we want to examine whether there are phenotype differences between Ndst4 KO and wild type (WT) mice. In this part, we used Southern blotting and multiplex PCR to identify the genotype of Ndst4 KO mice. Then, we used RT-PCR to check the tissue spectrum of Ndst4 RNA expression. We used the organ (Brain) with higher RNA expression to confirm that Ndst4 KO mice have no Ndst4 RNA expression. Finally, we conducted the modified-SHIRPA, complete blood count (CBC), biochemistry and histology analyses to compare Ndst4 KO and WT mice. In the modified-SHIRPA, CBC and biochemistry, we did not get significantly different results. However, in the histology analysis, we found that there were more goblet cells in proximal colon, and small intestine had morphological change in Ndst4 KO mice. Second, we want to produce mouse Ndst4 antisera. Because Ndst family has four members that have high similarity in protein sequences. So, we aligned all of the Ndst protein sequences by CLC sequence view 6 software, and found out a region with lower similarity. Then, we selected the sequences selected to perform B-epitope prediction. According to the information obtained, we decide to use 40 to 226 amino acids as immunogen. We used E coli system to express the target protein, and then performed protein purification. Finally, we used Balb/C mice to perform antigen immunization, and used Western blotting to determine titers and specificity of mouse antisera. All of the mouse antisera could recognize mouse Ndst4 antigen. In addition, 2 mouse antisera also could recognize human NDST4 (hNDST4) antigen. At present, we have got mouse antisera against mNdst4 and hNDST4 that could be applied at NDST4-associated study in the future.