CONSTRUCTION OF CD34 EXTRACELLULAR REGION ENCODING cDNA CLONE, AND EUKARYON EXPRESSION VECTOR AND ITS PRELIMINARY APPLICATION IN MONOCLONAL ANTIBODY PREPARATION BY GENE IMMUNIZATION
- VernacularTitle: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
- Author:
Yuying SUN
;
Yongzhi XI
;
Liying WANG
- Publication Type:Journal Article
- Keywords:
antigen, CD34;
hematopoietic stem/progenitor cells;
antibodies, monoclonal;
eukaryon expression vector, gene immunization
- From:
Medical Journal of Chinese People's Liberation Army
2001;0(12):-
- CountryChina
- Language:Chinese
-
Abstract:
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, 50?l 25% saccharin solution was injected into mouse musculus quadriceps femoris. Fifteen minutes later, blank control PBS, PBS diluted empty vector pcDNA3.1(50?g), 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.
