Zhou Fu-Ling, Zhang Wang-Gang, Meng Xin, Chen Gang, Wang Jian-Li
Department of Hematology, The Second Hospital, Xi'an Jiaotong University, Xi'an 710004, Shaanxi Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2008 Jun;16(3):466-71.
This study was aimed to investigate a novel MLAA-22 antigen derived from a U937 cDNA library by the SEREX approach and search for gene expression in various samples. Bioinformatic analysis was performed to forecast MLAA-22 information mined from databases and experimental datasets. CTL epitope was predictied and the specific antibody for MLAA-22 was elicited by using peptide-microspheres and adjuvants. Furthermore, SYBR Green real-time PCR and immunoblotting method were used to evaluate the specificity of gene expression. The results showed that the full length cDNA of MLAA-22 located on chromosome 17q11.2 was 2.0 kb in size, and a putative protein was approximately 72.4 kD for 631 amino acids. The MLAA-22 encoded a cancer/testis antigen in human, which is nonsecreting type, plasmosin, labile protein, hydrophilia, thermostability, and without signal peptide. Many motifs might related to growth, proliferation, differentiation, and apoptosis. Antigenic peptides was synthesized as the antigen with Fmoc/PyBOP method. Rabbits were immunized by injecting the synthetic peptide-KLH to obtain antibody and the immune sera analyzed with ELISA were 1:8000. SYBR Green real-time PCR and Western blot showed that MLAA-22 presented with a higher number of copy messages in M(5), lower in CML, but not in gastric carcinoma, renal carcinoma, LNCaP cell lines and normal adult tissues, etc. It is concluded that mlaa-22 is a novel acute monocytic leukemia-associated antigen gene and be extended to further discovery.
本研究旨在通过SEREX方法研究源自U937 cDNA文库的新型MLAA-22抗原，并在各种样本中寻找基因表达。进行生物信息学分析以预测从数据库和实验数据集中挖掘的MLAA-22信息。预测CTL表位，并使用肽微球和佐剂引发针对MLAA-22的特异性抗体。此外，使用SYBR Green实时PCR和免疫印迹法评估基因表达的特异性。结果表明，位于17q11.2染色体上的MLAA-22全长cDNA大小为2.0 kb，推测的蛋白质约72.4 kD，含631个氨基酸。MLAA-22在人类中编码一种癌/睾丸抗原，为非分泌型、血浆素、不稳定蛋白、亲水性、热稳定性且无信号肽。许多基序可能与生长、增殖、分化和凋亡有关。采用Fmoc/PyBOP方法合成抗原肽作为抗原。通过注射合成肽-KLH免疫兔子以获得抗体，ELISA分析的免疫血清为1:8000。SYBR Green实时PCR和蛋白质印迹显示，MLAA-22在M(5)中呈现较高的拷贝数，在慢性粒细胞白血病中较低，但在胃癌、肾癌、LNCaP细胞系和正常成人组织等中未检测到。结论是mlaa-22是一种新型急性单核细胞白血病相关抗原基因，有待进一步深入研究。
