Identification And Functional Studies Of T-cell Leukemia Related Novel Gene J441

Objective:To find and functionally identify the novel genes related to T-cell acute lymphoblastic leukemia.Methods:1.Sequencing was carried out with high-throughput technology for transcriptome profiling with T-cell acute lymphoblastic leukemia Jurkat cell. A new gene was discovered by bioinformatics comparative analysis upon the human genome data, designated as J441.2. J441 was identified in Jurkat cells by PCR.3. For further study, a plasmid contained the cDNA of FLAG-J441 fusion gene was constructed by PCR and packaged into pHAGE-CMV-MCS-Izs-Green lentivirus vector. The recombinant plasmid pHAGE-J441-FLAG was identified by PCR and sequencing. The expression vectors containing the green and red fluorescent protein were constructed at the same time.4. J441 was detected in malignant cell lines and tumor tissues by immunofluorescence and histopathological analysis.5. After infection of the recombinant lentivirus, the expression of J441 in 293T was investigated by the expression of ZsGreen and sequencing. The expression of FLAG was investigated by immunofluorescence.6. Bioinformatic prediction and analysis of J441 was performed through a variety of software and databases.Results:1. J441 is a novel gene mapped to chromosome 19. The protein encoded by J441 gene consisted of 183 amino acids, the formular of which was C897H1287N251O271S6. The isoelectric point was 6.51. The molecular weight was 20,215.76. The extinction coefficient was 52940 at 280nm. The estimated half-life is: 30 hours (mammalian reticulocytes, in vitro);>20 hours (yeast, in vivo);>10 hours (Escherichia coil, in vivo). Seven possible functional domains were found by IntrProScean.2. A recombinant lentivirus plasmid pHAGE-J441-FLAG was successfully constructed.The viral titer was 5×107 ifu/ml. The expression of J441 in 293T could be detected by the expression of ZsGreen and sequencing. Immunofluorescence showed that FLAG was expressed in cytoplasm.3. TLAP was highly expressed in Jurkat, Clone E6-1, HL60, cervical carcinoma, rhabdomyosarcoma, nasal-T cell lymphoma, hepatic carcinoma, metastatic carcinoma of the pancreas by histopathological analysis. While the expressions of J441 mRNA and protein were low, even absent, in normal tissues.Conclusion:J441 was high-level expression in malignant cell lines and tumor tissues. Bioinformatics analysis provided evidences for further study for the functions of J441 gene. We successfully constructed a lentivirus-based delivery system to stably express exogenous J441-FLAG The result of immunofluorescence suggested that the J441 gene might be located in cytoplasm. The new protein might be associated with tumorigenesis.