CAJ | 학술논문

目的了解不同细胞KEL基因转录本的差异.方法分别抽取K阴性、K阳性、K0的DNA、红系细胞RNA和总RNA,应用逆转录PCR、巢式PCR分别扩增、测序及克隆测序分析KEL基因第1～19外显子以及第2～8外显子.同时,4种单抗标记后流式细胞术检测红、白细胞上的Kell蛋白表达情况.结果红系RNA均为忠实于基因组序列的正常KEL转录本,而K0产生4种不同的转录本.但总RNA作模板时,可以发现不同个体产生不同的转录本,以正常、缺失第3外显子、第7外显子之前插入16 bp内含子的转录本为最常见,这些异常拼接也见于K0的红系RNA;总RNA第1～19外显子扩增片段虽然电泳条带只有一条,但测序显示为多种序列的混合.流式细胞技术检测证实K0红细胞上无Kell抗原的表达,其余个体的红细胞均有Kell抗原的强表达,但其白细胞上有不同程度Kell抗原的弱表达或无表达.结论 KEL基因在不同细胞中存在不同的转录本,可能导致Kell抗原在红、白细胞上有表达或无或弱表达.证实了KEL基因的表达转录研究中红系mRNA比总RNA更可靠.
목적 료해불동세포KEL기인전록본적차이.방법 분별추취K음성、K양성、K0적DNA、홍계세포RNA화총RNA,응용역전록PCR、소식PCR분별확증、측서급극륭측서분석KEL기인제1～19외현자이급제2～8외현자.동시,4충단항표기후류식세포술검측홍、백세포상적Kell단백표체정황.결과 홍계RNA균위충실우기인조서렬적정상KEL전록본,이K0산생4충불동적전록본.단총RNA작모판시,가이발현불동개체산생불동적전록본,이정상、결실제3외현자、제7외현자지전삽입16 bp내함자적전록본위최상견,저사이상병접야견우K0적홍계RNA;총RNA제1～19외현자확증편단수연전영조대지유일조,단측서현시위다충서렬적혼합.류식세포기술검측증실K0홍세포상무Kell항원적표체,기여개체적홍세포균유Kell항원적강표체,단기백세포상유불동정도Kell항원적약표체혹무표체.결론 KEL기인재불동세포중존재불동적전록본,가능도치Kell항원재홍、백세포상유표체혹무혹약표체.증실료KEL기인적표체전록연구중홍계mRNA비총RNA경가고.
Objective To investigate the difference of the transcripts between reticulocyte and non-reticulocyte cells in human blood. MethodsGenomic DNA, reticulocyte RNA and total RNA of K- , K+ and Kell-null(K0) were extracted, then PCR, reverse transcription-PCR(RT-PCR) and nested PCR followed by sequencing or cloning-sequencing were used to analyze the KEL gene rnRNA exons 1-19 and exons 2-8. Four kinds of monoclonal antibodies were labeled to detect the expression of Kell glycoprotein on red cells or leukocytes with flow cytometry. Results In reticulocyte,only one normal KEL transcript faithful to the genomic structure was found in all tested samples except K0 which had 4 different transcripts. Sequence analysis of exons 2-8 of total RNA confirmed the alternative KEL transcripts existed in different samples, mostly caused by abnormal splicing, among them, skipping of exon 3 and a 16 bp insertion of intron 6 at the beginning of exon 7 were the most frequent. Although only one band was observed after amplifying the exons 1-19 from total RNA, the sequencing result showed it was a mixture of different sequences. There was strong expression of Kell glycoprotein on red cells except K0, but no or low expression on leucocytes by flow cytometry. Conclusion Alternative transcripts of KEL gene exist in different cells, which would be responsible for different Kell glycoprotein expression patterns on different cells. This study suggested that reticuloeyte RNA was more suitable than total RNA for molecular study of KEL gene transcription.