先前研究中發現，罹患鼻部NK/T細胞淋巴癌 (human EBV-associated nasal natural killer/T-cell lymphoma) 的病人通常在染色體6q25.2-25.3這段區域中存在著染色體片段的缺失。其中最常見的是一段長度大約2.6 Mb的區域。這個研究結果顯示，這段區域中可能存在有某些與淋巴癌的生成相關的基因。C6orf35正是一個位在染色體6q25.3的新穎基因，到目前為止，這個基因還沒有任何相關的研究報告。從生物資訊資料庫中可得知，這個基因可能存在有兩個轉錄本（transcript）。為了瞭解這個基因在人體中表現的情形，我們設計了兩對具有專一性的引子（primer），並利用人類的cDNA　panel 做進一步的檢驗。實驗結果證明這兩個轉錄本確實表現在許多人類組織中。此外，我們也利用生物資訊工具對這兩個蛋白質的特性做分析，分析結果指出兩個蛋白質都具有穿膜性的結構（transmembrane domain）以及一個功能未知的蛋白質結構DUF1358 domain，而兩者最可能表現的位置都在細胞質。為了更進一步研究探討這兩個蛋白質，我們分別利用蛋白質N端及C端的胜肽序列設計了五支抗體。本研究的目的主要是針對這五支抗體在實驗應用上的專一性及靈敏度做測試，並運用這些抗體對C6orf35這個蛋白質的功能做進一步的研究。目前我們已經確認這五支抗體都能夠成功運用在西方墨點法（Western blot），免疫沉澱（Immunoprecipitation）以及免疫細胞染色（Immunocytochemistry）的實驗上。除此之外，對人類組織或一些細胞株做蛋白質表現檢測的部分，我們發現C6orf35L蛋白表現在許多人類組織中，其中又以在睪丸及卵巢中的表現量最大；值得一提的是，從這些受檢驗的組織中我們並沒有發現C6orf35S蛋白質存在的蹤跡。利用免疫沉澱以及串聯質譜檢測（tandem mass spectrometry analysis）等實驗，我們希望能了解C6orf35L蛋白質可能具有的功能。實驗結果顯示C6orf35L蛋白質可能會與熱休克蛋白70（HSP70）作用，有趣的是，它也可能會和自己形成多聚體的狀態。為了了解在轉錄層面的調控，我們也利用一些生物資訊工具做分析，並進一步設計實驗來驗證。利用HeLa細胞做低氧的處理測試，我們發現細胞在低氧的環境下，細胞中C6orf35L的訊息核醣核酸（mRNA）及蛋白質表現量的確有明顯下降；但是其訊息核醣核酸和正常情形下表現量並沒有統計上的差異。這個現象顯示C6orf35L有可能受到HIF1-α的調控。總而言之，這些研究結果確立了五支C6orf35專一性抗體的效力，也讓我們對為C6orf35基因的蛋白質層次做了較深入的了解，為將來進行相關的研究奠定一個更穩固的基礎。

The C6orf35 gene mapped to chromosome 6q25.3 region and encoded a novel, uncharacterized protein. As our previous work identified a 2.6 Mb common deletion interval on chromosome 6q25.2-q25.3 region was related to human EBV-associated nasal natural killer/T-cell lymphoma, it suggested that C6orf35 can be the candidate gene involved in the development of this type of lymphoma. Up to now, no report has been published for the study of C6orf35 gene. Two transcripts were predicted to be resulted from alternative splicing of C6orf35 DNA sequences. Using isoform-specific primers in commercial cDNA panels, we have previously confirmed the expressions of these two transcripts are ubiquitously in many human tissues. The predicted characteris-tics using bioinformatics analyses suggested both transcripts have transmem-brane domains, a DUF1358 domain, and may locate in the cytoplasm. To illu-strate the function of C6orf35, this study aims to generated antibodies that recognize both isoforms and used for further investigation. We have generated 5 antibodies that cover both N- and C-terminals of the deduced C6orf35 pro-teins and tested the specificity and sensitivity of these antibodies in Western blot, immunoprecipitation and immunocytochemistry analyses. Using the antibodies, the expression of C6orf35 protein in human tissues and cell lines was determined. The results showed while C6orf35L protein expresses in many human tissues and cell lines, with the most abundant expression in testis and ovary, C6orf35S protein was not detected in any of the tissues examined. Results from immunohistochemistry showed that C6orf35L protein expressed in almost all cell types in human adult testis and colon tissue. To identify the potential interacting protein of C6orf35L, co-immunoprecipitation was con-ducted and heat shock protein 70 (HSP70) and C6orf35L itself were identified by using tandem mass spectrometry analyses. In addition, we found that the expression of C6orf35L mRNA and protein were reduced under hypoxia treatment thus the data suggests the HIF1-α may be involved in the regulation of C6orf35L. Results from this study not only provide the information of ef-ficacy of C6orf35 antibodies but also improve our understanding about the characteristics of C6orf35.

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