Optimization Of Human TRIM5α Chimera Expression In E.coli And Studying The Mechanism Of Its' Inhibition To HIV-1

Study BackgroundIn 2004, TRIM5a was screened from rhesus monkeys, which is a cytoplasm factor can inhibit to the HIV-1, Rhesus monkeys TRIM5a and that of macaques can inhibit to HIV-1, but not SIVmac, on the contrary, human TRIM5a can't inhibit to those viruses, has the ability of inhibition to the Y-retrovirus N-MLV. However, when several amino acids change, the human TRIM5 alpha will have similar level of anti-HIV-1 as the Rhesus TRIM5a. In addition, because of the immune response of human has anti-Heterogeneous animal, heterologous protein in the human body is often quick to be removed, and the half-life is shorter, so human TRIM5a(mutant) was chosen for studying in our laboratory.But in the past, the majority of the researches about TRIM5a were finished by gene transfection, For example, TRIM5a gene was conveyed into the cells by liposome or viral vector, but there are many problems during the operative procedure, such as poor feasibility, low efficiency and stability, low transfection rate, biological toxicity, so the E.coil expression systems of PTD-TRIM5a and TRIM5aH (R328-332) were constructed early in our lab, but the efficiency of it were not high.The transmembrane efficientcy, cytotoxicity to target cells and inhibition to HIV-1 of PTD-TRIM5a and TRIM5aH (R328-332) were researched before, shows that PTD-TRIM5a and TRIM5aH (R328-332) can entry into cells, can play biological effect on target cells and have no toxicity. Although PTD-TRIM5a and TRIM5aH (R328-332) are from human, several amino acids were replaced by our lab, the safety of it is necessary to study for next, especially for normal human cells.So far, the mechanism of TRIM5a inhibition to HIV-1 has become more and more clear, however, the reaction of TRIM5a restricted HIV-1 replication is not yet entirely clear, many questions need to be resolved, most remain at the level of speculation, In our lab, TRIM5a can bind with gag of HIV-1 capsid protein directly has studied, but it will result in reposition, modification or degradation of nucleocapsid also need to research. Whether TRIM5a contains a ubiquitin ligase subunit, or a subunit which is similar with small ubiquitin-related modifier, the process of reverse is blocked by TRIM5a, or synthesized DNA were degraded, or other proteins were combined with TRIM5a, all of this will remain to study.Objective:1. The recombinant plasmid of pET28a previously constructed was transformed into colorectal strain BL21 (DE3), testing PTD-TRIM5a and TRIM5aH (R328-332) were expressed on level of gene and protein.2. To optimize the expression of human TRIM5a chimera protein in E. coli DE3, we have compared with expression product of human TRIM5a Recombinant Plasmid which was constructed in our lab in different conditions.3. Although PTD-TRIM5a and TRIM5aH (R328-332) are from human, several amino acids were replaced by our lab, the safety of it is necessary to study for next, cytotoxicity of normal human cells were researched in this study.4. In our lab, TRIM5a can bind with gag of HIV-1 capsid protein directly has studied, in order to study the mechanism of TRIM5a inhibition to HIV-1, Subcellular localization of TRIM5a inhibition to HIV-1 was analyzed by confocal laser screening microscopy.5. To be defined the relationship of ubiquitination and capacity of TRIM5a.Methods1. Using enzyme digestion, PCR, gene sequencing on the gene level, and SDS-PAGE, Western-blotting, peptide mass fingerprint (PMF) analysis, to test the expression of PTD-TRIM5a and PTD-TRIM5aH (R328-332).2. Protein products which expressed in different temperature, concentration of IPTG, duration of induce, induce time, medium were analyzed by SDS-PAGE, maximal expression of each condition was found out separately.3. Cytotoxicity of PTD-TRIM5a and PTD-TRIM5aH (R328-332) to normal human cells was studied by trypanblau and SunBioTMAm-Blue.4. Recombinant protein and nucleus were labeled with FITC and Hoechst separately, the distribution of fluorescence was observed under confocal laser screening microscope, Subcellular localization of TRIM5a inhibition to HIV-1 was analyzed.5. To be defined the relationship of ubiquitination and capacity of TRIM5a by ELISA.Results1. Target bands or peaks were appeared on level of gene and protein, so PTD-TRIM5a and PTD-TRIM5aH (R328-332) had expressed.2. Through analysis and comparison, maximal expression of recombinant plasmid was induced by 0.5mmol/L IPTG at 30℃for 8 h when OD value of bacterium was 0.6.3. Recombinant protein was added into sample,83% 3T3 cells were survived, so the recombinant protein was safe.4. Analysis of the color of fluorescent, shows that the recombinant protein is only limited into the cytoplasm, not enter nucleus.5. when concentration of PTD-TRIM5a and PTD-TRIM5aH(R328-332) were 100μg-mL-110μg-mL-1 1μgmL-1,0.1μg-mL-1,0.01μg-mL"1 respectively, its' concentration of UBPL were about 225.7pg-mL-1,160 pg-mL-1,125.7 pg-mL-1,57.1 pg-mL,41.4 pg-mL-1 and 720 pg-mL-1,304.3 pg-mL-1,164.3 pg-mL-1,88.6 pg-mL-1,51.4 pg-mL-1 separately.Conclusions1. As effort of our lab, prokaryotic expression systems of E.coli of PTD-TRIM5a and PTD-TRIM5aH(R328-332) were successfully constructed, it was identified on gene and protein level.2. After the optimization of individual condition factor, recombinant protein expression level significantly enhanced after the optimal conditions.3. Although PTD-TRIM5a and TRIM5aH (R328-332) are from human, several amino acids were replaced by our lab, the safety of it is necessary to study for next, cytotoxicity of normal human cells were researched, and it is safe.4. Analysis of the color of fluorescent, shows that the recombinant protein is only limited into the cytoplasm, not enter nucleus, so recombinant protein can not entry into nucleus for inhibiting to replication of HIV-1.5. From the test of ELISA, a certain linear relationship was showed between ubiquitination of recombinant protein and ability of anti-HIV-1 of it.