摘要
ＣＤ３４分子是高度糖基化的Ⅰ型跨膜蛋白，主要表达于多功能造血干细胞、祖细胞表面，在成熟血细胞表面无ＣＤ３４抗原表达，提示ＣＤ３４分子在早期造血调控方面起着重要的作用。本文采用ＲＴ－ＰＣＲ方法，从高表达人ＣＤ３４抗原的ＫＧ－１ａ细胞系中成功地克隆出人ＣＤ３４抗原全长ｃＤＮＡ，并将此基因插入克隆“载体ｐＵＣ１８ＨｉｎｃＩＩ酶切位点，构建了重组质粒ｐＵＣ１８－３４。用双酶切ｐＵＣ１８－３４质粒，将分离得到的基因片段插入痘苗病毒载体的ＳｍａⅠ位点，构建了重组质粒ＰＪＳＡ１１７５－３４。采用Ｌｉｐｏｆｅｃｔｉｎ方法，ＰＪＳＡ１１７５－３４转染已被野生痘苗病毒感染的ＴＫ￣－１４３细胞，用ＢｕｄＲ和ＬａｃＺ双筛选，获得带有人ＣＤ３４抗原全长ｃＤＮＡ的重组痘苗病毒。经活细胞荧光染色和ＡＰＡＡＰ检测，表明重组痘苗病毒能特异地表达人ＣＤ３４抗原。人ＣＤ３４抗原ｃＤＮＡ克隆和表达，为进一步研究其结构和功能的关系以及研究造血调控机理奠定了基础。
he
human CD34 surface antigen,which is a type Ⅰ transmembrane
glycoprotein with noobvious homology to other known proteins, is
selectively expressed on hematopoietic stem andprogenitor
cells,suggesting that it plays an essential role in early
hematopoiesis. Using gene spe-cific primers, reverse
transcription,and polymerase chain reaction methods. We isolated a
full-length CD34 cDNA from KG-1a cell line which high expresses CD34
antigen. We inserted thefull-length CD34 cDNA into Hinc Ⅱ enzyme
site of pUC18 plasmids to construct recombinantplasmids pUC18-34.
Expression vectors named PJSA1175-34 was constructed by inserting
theCD34 cDNA, under the control of the promoter 7.5k,into the TK gene
region of vaccinia virusvectors of Tian Tan strain, Using the
lipofectin method and double selection of BudR and Lac Z,TK￣-143
cells infected with the derived recombinant plasmids PJSA1175-34 in
the present of in-fectious TK￣+ vaccinia virus yielded recombinant
vaccinia virus.By IFA and APAAP methods,CD34 antigen was detected in
the surface of the infected cells. The clone and expression of
thehuman CD34 gene should faciliate the study of the function and
regulation of the stem cell sur-face antigen.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
1995年第2期79-81,共3页
Chinese Journal of Immunology
