人CD34抗原胞外区cDNA的克隆、真核表达载体的构建及在基因免疫制备CD34单克隆抗体中的初步应用  

CONSTRUCTION OF CD34 EXTRACELLULAR REGION ENCODING cDNA CLONE, AND EUKARYON EXPRESSION VECTOR AND ITS PRELIMINARY APPLICATION IN MONOCLONAL ANTIBODY PREPARATION BY GENE IMMUNIZATION

作　　者：孙玉英[1] 奚永志[1] 王丽英[1] 崔建武[1] 刘楠[1] 梁飞[1] 

机构地区：[1]解放军第307医院,北京100039

出　　处：《解放军医学杂志》2003年第12期1090-1092,1095,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金资助课题 (编号 39370 2 72 )

摘　　要：目的　克隆含编码人CD34抗原胞外区的cDNA ,并构建其真核表达载体 ,探讨基因免疫制备抗人CD34单克隆抗体的可行性。方法　从KG 1a细胞提取总RNA ,RT PCR扩增含编码人CD34抗原胞外区的cDNA并酶切测序鉴定 ,构建其真核表达载体pcD NA3 1 CD34。选取 4～ 6周龄的BALB/c小鼠 12只 ,随机分为 3组 ,预先在股四头肌注射 2 5 %的蔗糖溶液 5 0 μl,然后在相同部位分别注射PBS空白对照、PBS稀释的空载体pcDNA3 1以及PBS稀释的pcDNA3 1 CD34,每 2周 1次共 3次。免疫结束后定期FACS检测鼠尾静脉血抗人CD34抗体产生情况。结果　所扩增的人CD34抗原胞外区cDNA全长 886bp ,扩增片段大小与理论值相符 ,测序结果与文献报道完全一致 ,并且在 5′端引入HindⅢ酶切位点 ,3′端引入EcoRI酶切位点及终止密码TGA。随后将其定向插入真核表达载体 pCD NA3 1中 ,称之为pcDNA3 1 CD34,经阳性克隆筛选、酶切鉴定证实 pcDNA3 1 CD34真核表达载体构建成功。FACS检测结果表明 ,用pcDNA3 1 CD34真核表达载体免疫的小鼠中仅 1/ 4只在免疫结束后的第 2～ 6周有较低滴度的CD34抗体产生 ,其余 3只血清中均未检测到CD34抗体。结论　成功地构建了人CD34抗原胞外区cDNA的真核表达载体 pcDNA3 1 CD34。Objective To clone CD34 extracellular region encoding cDNA and to construct its eukaryon expression vector to explore the feasibility of its monoclonal antibody preparation by gene immunization. Methods Total RNA was extracted from KG-1a cell lines. CD34 extracellular region encoding genes were amplified by RT-PCR method and then confirmed by enzymatic lysis and DNA sequencing, and its eukaryon expression vector was constructed as pcDNA3.1-CD34. Twelve BABL/c mice aged 4-6 weeks were selected and randomly divided into 3 groups. Before immunization, 50μl 25% saccharin solution was injected into mouse musculus quadriceps femoris. Fifteen minutes later, blank control PBS, PBS diluted empty vector pcDNA3.1(50μg), or PBS diluted pcDNA3.1-CD34 was injected into the same site of the above three groups, respectively. Immunization was taken every two weeks for a total of three times. The antibody was detected regularly by FACS using tail blood from immunized mice. Results The results demonstrated that the length of CD34 cDNA was 886bp which was identical to the theoretical value and its sequence was confirmed by DNA sequencing which was identical to the registered sequence. The accuracy of CD34 expression vector recombination was confirmed by restriction enzymatic lysis. Hind III and EcoRI restriction enzymatic lysis sites were introduced into 5 and 3 terminals of amplified cDNA sequence, respectively. Terminate code TGA was also introduced into CD34 extracellular encoding cDNA. The expression vector possessing target genes was named as pcDNA3.1-CD34. FACS detection indicated that only 1/4(25%) immunized mice had a lower titer CD34 antibody in their tail vein serum 2-6 weeks after immunization, and the others did not show no antibody production. Conclusion The eukaryon expression vector pcDNA3.1-CD34, which express CD34 extracellular region, has been constructed. The feasibility of CD34 McAb preparation can primarily be confirmed by gene immunization.

关 键 词：抗原 CD34 造血干/祖细胞 抗体 单克隆 真核表达载体 基因免疫 

分 类 号：Q782[生物学—分子生物学]