Dissertation
Dissertation > Medicine, health > Basic Medical > Medical Immunology

Study on the Interactions of Human T Cell Immunoglobin Mucins with Apoptotic Cells

Author ChenZhiZhong
Tutor HuLiHua
School Huazhong University of Science and Technology
Course Clinical Laboratory Science
Keywords TIM-1 TIM-3 RT-PCR SYBR GreenⅠ gene expression mRNA TIM-4 Bioinformatics Analysis Clone Fusion protein Apoptosis T cell immunoglobulin mucin gene fusion protein receptor immune response apopotosis pervanadate E coli
CLC R392
Type PhD thesis
Year 2011
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PartⅠEstablishment of a real-time SYBR GreenⅠquantitative RT-PCR assay for detection of human TIM-1 and TIM-3 mRNAObjective:This study aimed to develop a SYBR GreenⅠreal-time quantitative RT-PCR assay (FQ-PCR) for detection of human TIM-1 and TIM-3 mRNA.Methods:Total RNA extracted from peripheral blood mononuclear cell (PBMC) of human was reverse transcribed to cDNA. The prospective amplicons of human TIM-1 and TIM-3 were amplified and purified, then were ligated with pMD18-T Simple vector and transformed into bacterium DH5a, respectively. Plasmid DNA extracted from positive clones were verified by PCR amplification and sequenced. The concentration of purified DNA template was detected by analyzing absorbance in 260 nm and then the combined plasmid was diluted to series as standard for FQ-PCR. TIM-1 and TIM-3 were amplified by real-time fluorescence quantitative PCR from the plasmid DNA, respectively.Results:The methods of TIM-1 and TIM-3 mRNA real-time PCR were well established, which detected as low as 103 copies with the linear range from 103 to 107 copies. The standard curves showed that there were good linear relationships between Ct value and the logarithm of their concentrations, the correlation coefficients of TIM-1 and TIM-3 were 1.00 and 1.00, respectively. The PCR efficiencies were 1.070 and 1.023, respectively. Intra-assay and inter-assay coefficiency variations for both genes were less than 5%.The melting curve showed a single peak. Conclusions:We have been establish successfully a SYBR Green I real-time quantitative RT-PCR assay (FQ-PCR) for detection of human TIM-1 and TIM-3 mRNA, which will provide useful methodological basis for understanding functions of human TIM-1 and TIM-3 genes. Part II Bioinformatics analysis and construction of eukaryotic expression vector of human TIM genes and theirs fusion proteinObjective:To construct respectively eukaryotic expression vectors of human TIM genes and theirs fusion protein. This study also aimed to conduct bioinformatics analysis of human TIM genes.Methods:The experiment extracted total RNA from human peripheral blood monouclear cells (PMBC), and amplified three members of human TIM family and theirs ectodomain as well as IgGl Fc gene fragment with two steps RT-PCR technique. They were respectively cloned into the eukaryotic expression vector pcDNA3.1 (+), confirmed by PCR and sequencing. Then the ectodomain of human TIM genes was subcloned into the eukaryote expression vector pcDNA3.1 (+) which contained the fragment of human IgGl Fc gene after sequence analysis, and identified by PCR combined with double restriction enzyme digestion. The physicochemical characteristics, structures and functions of TIM genes were predicted and analyzed with means of bioinformatics.Results:Evidence of DNA sequence analysis, PCR and restriction enzymes digestion showed that the fragments were the same as the sequences provided by GenBank. The human TIM genes and theirs fusion proteins were constructed were successfully cloned into the eukaryotic expression vector pcDNA3.1(+).The results of bioinformatics analysis suggested TIM-1 protein was a unstable hydrophilic protein, containing one transmembrane domain, with a relative molecular weight of 39.2KD and isoelectric point of 6.44. The protein include a-helix (23.08%), Extended strand (29.67%), random coil (47.25%) and a signal peptide of 20 amino acid residues. Its subcellular localization was in endoplasmic reticulum, Golgi and plasma membrane. Function analysis predicted TIM-1 have functions in receptor, signal transduction and immune response.TIM-3 protein was a stable hydrophilic protein, containing one transmembrane domain, with a relative molecular weight of 33.4KD and isoelectric point of 5.54. The protein include a-helix (32.56%), Extended strand (8.27%), random coil (49.17%) and a signal peptide of 21 amino acid residues. Its subcellular localization was in endoplasmic reticulum, vacuolar, Golgi and plasma membrane. Function analysis predicted TIM-3 have functions in receptor and signal transduction.TIM-4 protein was a stable hydrophilic protein, containing one transmembrane domain, with a relative molecular weight of 41.6KD and isoelectric point of 5.75. The protein include a-helix (10.32%), Extended strand (29.89%), random coil (59.79%) and a signal peptide of 24 amino acid residues. Its subcellular localization was mainly in cytoplasm, nucleolus, vesicles of secretory system, mitochondria, endoplasmic reticulum and golgi. Function analysis predicted TIM-4 have functions in receptor and signal transduction.Conclusions:The human TIM genes and theirs fusion protein are constructed successfully and the bioinformatics analysis is made by biological analysis software to investigate theirs character. It provides foundation for further study. PartⅢStudy on the interactions of human T cell immunoglobin mucins with apoptotic cellsObjective:To study the interactions of human TIM family with apoptotic cells and further explore the roles of human TIM genes in apoptosis.Methods:We constructed nine kinds of pEGFP-Nl eukaryotic expression vector containing different length of the three members of human TIM genes and the expression vectors of theirs Ig fusion proteins.Results:We found that human TIM proteins recognize and bind to apoptotic cells directly, but not to viable cells. The interactions of sTIM-1-EGFP, sTIM-3-EGFP and sTIM-4-EGFP with apoptotic cells are blocked by TIM-1-Ig, TIM-3-Ig and TIM-4-Ig fusion proteins, respectively. The results also suggest that human TIM proteins mediate the recognition of apoptotic cells and bind to apoptotic cells directly via IgV domains.Conclusions:Our results show that human TIM proteins may act as receptors of apoptotic cells and play a key role in the regulation of apopotosis. These data also indicate that human TIM proteins may serve as novel proteins to detect apoptosis. PartⅣConstruction and characterization of human TIM-EGFP fusion proteins in Escherichia coliObjective:The study aims to explore the expression and purification of a fusion protein of human TIM genes and EGFP in E. coli and evaluate theirs bioactivity.Methods:The DNA fragments of three members of human TIM genes and EGFP genes were respectively amplified by PCR and cloned into prokaryotic expression vector pET-28a. The constructed recombinant plasmids containing pET-28a-TIM-EGFP were respectively transformed to E.coli BL21 (DE3) for the expression under the induction of IPTG. The expressed proteins were respectively purified by Ni-NTA resin and theirs binding activity to the apoptotic cells were detected under the fluorescence microscope.Results:We successfully constructed the TIM-1-EGFP, TIM-3-EGFP and TIM-4-EGFP vectors and expressed them in E coli, respectively. Our results demonstrated that all three TIM-EGFP fusion proteins recognize and bind directly to apoptotic cells, but not to viable cells. We further verified that the interactions of TIM-EGFP with apoptotic cells were blocked by TIM-Ig fusion proteins, respectively.Conclusions:We successfully constructed the TIM-1-EGFP, TIM-3-EGFP and TIM-4-EGFP vectors and expressed them in E coli, respectively. To our knowledge, it is the first time that three members of human TIM genes are overexpressed in E. coli till date. Our results have also demonstrated that human TIM proteins mediate the recognition and binding of apoptotic cells. Furthermore, the fusion protein shows a readily obtainable source of biologically active TIM-1、TIM-3 and TIM-4, which has considerable potential for further studies on human TIM genes and their receptors.

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