hsFLJ22386基因位於染色體16p13.3位置上，此基因的蛋白質產物含有287個胺基酸，分子量大約為32.2 kDa。經由生物資訊程式預測hsFLJ22386的二級結構，發現其可能是一個含有leucine zipper domain的蛋白。根據RT- PCR實驗的結果顯示，FLJ22386在腦神經組織及肝、脾、小腸、腎等器官組織有大小不同的表現。而在人類細胞株有表現FLJ22386基因的有腦瘤細胞（T98G、U87MG、U251、GBM 8401）、鼻咽上皮細胞（NNE-3）、鼻咽癌細胞（NPCTW01、NPC TW04）、肝癌細胞（J5、Hep3B、SK-Hep-1）、淋巴癌細胞（RPMI、P3HR1、Raji、U937）有大小不同的表現。我也將人類FLJ22386基因嵌入pEGFP-C2載體，經由轉化作用使其在NIH 3T3細胞表現，觀察其是否有促進分裂增殖的能力。為了進一步研究FLJ22386蛋白層次的表現，我製作能辨識FLJ22386蛋白質的多株抗體，最後證明這多株抗體並沒有辦法辨識到想要的FLJ22386蛋白，而這抗體所辨識到的蛋白也還需要進一步釐清。綜合RT-PCR與anchorage dependent growth assay的結果，可以推測FLJ22386蛋白可能在細胞增殖扮演一個角色。

The hsFLJ22386 gene, an unknown gene, was located on chromosome 16p13.3. Its protein product contains 287 amino acids with the molecular weight of 32.2 kDa approximately. Predicted by bioinformatics, hsFLJ22386 might be a protein containing a leucine zipper domain. Based on the results of reverse transcriptase polymerase chain reaction, it revealed that FLJ22386 was expressed in several nervous system tissues and several organ tissues (liver, spleen, small intestine and kidney). In human cancer cell lines, the RT-PCR results showed that FLJ22386 was expressed in brain tumor cell lines (T98G, U87MG, U251, GBM8401), nasopharyngeal epithelial cell line (NNE-3)and carcinoma cell lines (NPCTW01, NPCTW04), hepatoma cell lines (J5, Hep3B, SK-Hep-1) and lymphoma cell lines (RPMI, P3HR1, Raji, U937). Human FLJ22386 coding sequence was inserted into pEGFP-C2 plasmid, and the tag-fused gene was transfected into NIH3T3 cells to see if it has the ability to promote cell proliferation. To further investigate the protein level expression and biological functions of the gene, glutathione S-transferase-hsFLJ22386 fusion protein was expressed and used to generate anti-FLJ22386 polyclonal antibody. According to the results of RT-PCR and anchorage dependent growth assay, it is presumed that FLJ22386 may play a role in cell proliferation.

中文摘要--------------------------------------------------1
英文摘要--------------------------------------------------2
前言------------------------------------------------------3
實驗目的-------------------------------------------------13
實驗材料與方法-------------------------------------------14
一、 細胞培養--------------------------------------14
二、 檢體收集--------------------------------------14
三、 反轉錄聚合酵素連鎖反應----------------------------14
四、 重組DNA質體之建構-------------------------------17
五、 細胞轉化作用--------------------------------------21
六、 生長曲線測試--------------------------------------24
七、 Anchorage dependent growth assay ---------------------25
八、 蛋白質表現及萃取----------------------------------26
九、 FLJ22386多株抗體製備 -----------------------------31
十、 以ELISA的方式測定抗體力價 -----------------------32
十一、 多株抗體IgG純化-------------------------------33
十二、 西方點墨法-------------------------------------35
十三、 In gel digestion ----------------------------------38
十四、 MALDI/TOF分析 -------------------------------40
實驗結果與討論-------------------------------------------41
一、 hsFLJ22386在各癌細胞株的表現----------------------41
二、 FLJ22386在成熟小鼠各器官組織的mRNA表現---------42
三、 生長曲線測試--------------------------------------42
四、 Anchorage dependent growth assay ---------------------43
五、 表現GST-FLJ22386蛋白-----------------------------43
六、 Anti-FLJ22386多株抗體製備 ------------------------45
七、 Anti-FLJ22386多株抗體IgG純化 --------------------45
八、 抗體與抗原親和性測試------------------------------46
九、 以cell line protein lysate測試抗體是否能辨識真正的hsFLJ22386----------------------------------------47
十、 hsFLJ22386在細胞內表現位置------------------------48
十一、 hsFLJ22386在乳癌組織與非癌化乳房組織的表現----48
十二、 MALDI-TOF/MS分析----------------------------49
結論-----------------------------------------------------51
參照網站-------------------------------------------------52
參考文獻-------------------------------------------------53
圖表附錄
圖一、利用RT-PCR檢測各細胞株hsFLJ22386 mRNA的表現情形--------------------------------------------------57
圖二、利用RT-PCR檢測各小鼠組織FLJ22386 mRNA的表現情形--------------------------------------------------58
圖三、pEGFP-C2/NIH3T3與pEGFP-C2/FLJ22386/NIH3T3這兩種stable clone的生長曲線測試 --------------------------59
圖四、Anchorage dependent growth assay---------------------60
圖五、蛋白質表現載體pGEX-6p-1--------------------------61
圖六、建構蛋白質表現載體pGEX-6p-1/FLJ22386------------62
圖七、pGEX-6p-1/FLJ22386/BL21在不同表現時間下之小量蛋白質表現 ---------------------------------------------63
圖八、利用GST親和性管柱純化系統純化GST-FLJ22386融合蛋白結果---------------------------------------------64
圖九、利用ELISA的方式測定anti-FLJ22386多株抗體血清的抗體力價---------------------------------------------65
圖十、利用Protein G beads column做抗體純化之後跑SDS-PAGE的結果 -------------------------------------------66
圖十一、抗體與抗原的親和性測試---------------------------67
圖十二、利用cell line及胎盤的total protein lysate做Western blot的結果 ---------------------------------------------68
圖十三、以Western blot檢測hsFLJ22386在細胞內表現的位置---69
圖十四、人類乳癌組織及非癌化乳房組織的FLJ22386蛋白表現量比較-----------------------------------------------70
圖十五、MALDI-TOF/MS分析結果------------------------71
圖十六、ENSEMBL網站對於hsFLJ22386基因的exon預測----72
圖十七、hsFLJ22386基因在染色體的位置--------------------73
圖十八、hsFLJ22386的蛋白序列及二級結構預測--------------74
圖十九、SOSUI及PSORTII網站對於hsFLJ22386蛋白的預測---75
圖二十、EMBL GFP-cDNA locaisation project所做的有關FLJ22386蛋白在細胞內表現區域-----------------------------76
圖二十一、FLJ22386其他物種的同源基因預測----------------77
圖二十二、SOURCE網站所預測hsFLJ22386在各組織mRNA的表現量排名-----------------------------------------78
圖二十三、CGAP所預測hsFLJ22386在不同器官中，正常組織與癌組織的mRNA表現量比較----------------------------79
圖二十四、屬於zipper domain的兩種protein-DNA complex模式-80
表一、PCR primers序列-----------------------------------81
表二、PCR condition-------------------------------------82
表三、MALDI-TOF/MS預測結果-------------------------- 83
表四、不同物種與FLJ22386同功蛋白的長度與相似度 ---------84
表五、NCBI資料庫裡與人類FLJ22386基因相關的cDNA序列—85
簡稱與全名對照表-----------------------------------------86
藥品與廠商對照表-----------------------------------------87
儀器與廠商對照表-----------------------------------------89

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