| BackgroudsEndotoxemia is a series of pathophysiological responses caused by toxins which are released by gram-negative bacteria in the body. It is also the main cause of systemic inflammatory response syndrome (SIRS) and multiple organ failure (MOF). The main component of the endotoxin, lipopolysaccharide (LPS) derived from the outer membrane of Gram-negative bacteria cell wall is composed by the core oligosaccharide, specific polysaccharide and lipid A which is the active site and the center virulence of LPS. LPS mediates almost all biological effects of endotoxin, activates the body’s immune response, and then makes the body produce various inflammatory mediators which trigger the body’s systemic inflammatory response and affect the integrity of the barrier function and organ function.It will cause febrile reactions, leukocyte reaction, endotoxemia and endotoxin shock.Endotoxemia, a common clinical critical illness, caused by serious infections is one of the main causes of death and inflammatory respone in newborns and children.Its high morbidity and mortality is a tough problem in the medical field. There is no other effective treatment except for the antibiotics and hormone. Antibiotics can control bacteremia effectively by inhibiting and killing bacteria, while large amounts of endotoxin would be released which causes a much higher level of endotoxin in plasma or body fluids. The significant quantity of endotoxin will activate excessive immune response and deteriorate the condition of endotoxemia.Thioredoxin (Trx), also known as interleukin-1-like cytokines, adult T-cell leukemia-derived factor,or early pregnancy factor, first discovered in1964in E. coli and1989in human body is a small molecules protein widely found in both prokaryotes and eukaryotes. Its active site is-Cys-Gly-Pro-Cys-which can catalyze oxidation-reduction reaction reversibly through the unique disulfide active center. According to positioning,there are three types of Trx-Trxl, Trx2and Trx3. Trxl is located in the cytoplasm, while Trx2in the mitochondria, and Trx3mainly in the sperm cells.Trx-1, the most extensively studied in the thioredoxin family is a multifunctional protein distributed both in intracellular and extracellular. In addition to a basic antioxidant function, it also has growth-promoting, anti-apoptotic. anti-inflammatory and regulation of transcription factor activity effects. In recent years, studies have found that human Trx-1is closely related to inflammatory disease. More research focus on its anti-inflammatory role and relative mechanism. Human Trx-1is excreted by kidney, the kidney concentration is ranked first.While the lung concentration of Trx-1is ranked second suggesting that Trx-1may be a key treatment for lung disease.ObjectsPrepare recombinant human thioredoxin1and antiserum, then detect the antiserum titer and specificity to verify its availability.In order to study whether recombinant human thioredoxin protein1has a protective effect to neonatal rats with endotoxin or not, the newborn rat model of endotoxemia was established and then intervented by the protein. It may lay the foundation for the further experiments.Methods1. acquisition of rhTrx-1cDNA and primers design:The total RNA was extracted from human fetal hepatocytes, and then transformed into cDNA by RT-PCR. Upstream and downstream primers were designed and synthesized according to the gene sequence of hTrx-1in the GenBank with the aid of DNA Club software. With the specific primers and high-fidelity DNA polymerase applied, PCR product was detected by agarose gel electrophoresis.2. construction and identification of the prokaryotic expression vector:PCR product, digested by Sall and SacI, were ligated with PET-28a (+) vector digested by the same restriction enzyme. Then the recombinant plasmid was transfected to Escherichia coli DH5a. Positive clones were selected by kanamycin. The extracted recombinant plasmid was identified by restriction endonuclease analysis and DNA sequencing.3. induction of recombinant protein:The extracted recombinant plasmid was transfected to Escherichia coli BL21.The positive clones were selected by kanamycin. The single colony was cultured in liquid LB with kanamycin all night and then added IPTG to a final concentration of1mmol/1to induce the expression of recombinant protein.Bacteria was collected by centrifugal separation.SDS-PAGE was performed to detect the purification and molecular weight of objective protein.4. expression and purification of recombinant protein:Based on the above experiment, the optimal condition of protein expression was determined, then a large number of the recombinant protein was induced. Bacteria was collected by centrifugal separation.Bacterial precipitation was resuspended with1x Buffer and then placed at4℃overnight.The next day, the supernatant and precipitation samples were respectively taken to perform SDS-PAGE to determine the solubility of recombinant protein after unfreezing and lysis by ultrasonic wave. It was expressed in the supernatant, then purified after the removal of endotoxin with His ion column.It was then taken to dialysis. Finally, the protein was taken to measure concentration using Bradford and stored at-80℃.5. preparation and identification of polyclonal antibody:Rats were first vaccinated subcutaneously with200μg of purified protein rhTrx-1mixed with the same amount of complete Freund’s adjutvant respectively,and then boosted twice,at two weeks intervals, with100μg of purified protein rhTrx-1mixed with the same amount of incomplete Freund’s adjutvant. Others received adjutvant mixed with PBS as a control group.Another received PBS alone without adjutvant as a negative control group. Blood was collected from rat tail before each vaccination and two weeks after the last Vaccination.The titer of antiserum was detected by ELISA. Western blot analysis was performed to detect the availability and specificity of polyclonal antibody.6. preliminary observations of the protective effect to neonatal rats with endotoxemia:We prepared animal model of endotoxemia using newborn rats.Thirty-six full-term newborn Sprague-Dawley rats aged senven days were randomly divided into three groups-experimental group, control group and negative control group. Each group was twelve.The rats of experimental group were received intraperitoneal injection with rhTrx-110milligram per kilogramn, and then with LPS5milligram per kilogram in the same way half an hour later, while control group were received intraperitoneal injection with LPS5milligram per kilogram, negative control group with0.9%saline0.1mL.Their general state and mortality of24hour were observed.The lung morphology of2hour and8hour was observed by hematoxylin-eosin stain.7. data analysis:Data statistics was done with SPSS13.0.Twenty-four hour mortality of three groups were compared using Chi-square test.P<0.05was selected as significant standard.Results1. identification of recombinant plasmid:The prokaryotic recombinant plasmid was dentified by PCR, restriction enzyme digestion and sequencing. Agarose gel electrophoresis was performed to detect enzyme digestion products.The result showed that there was a clear band with the molecular weight of approximately600bp. In addition, sequence analysis showed that the insertion fragment of recombinant plasmid was complete and the reading frame was correct.That proved the success of construction of recombinant plasmid.2. optimal induction time:With the extension of time, protein expression was gradually increased after adding IPTG inducer, but there was no significant change5hours later.3. protein expression and purification result:The recombinant plasmid was transformed into E. coli BL21. According to the molecular mass standards, rhTrx-1was27.9kDa consistent with the target protein following the SDS-PAGE identification. This protein mainly existed in the supernatant and could be purified by ion column which proved that the preparation and purification of rhTrx-1was successful.4. titer detection:The valeney of antiserum was shown by ELISA as1:51200after the last vaccination.5. Western blot: Western blot was performed, the immune serum reacted with purified recombinant rhTrx-1showing a single clear band, but normal rat serum reacted with purified recombinant rhTrx-1showing no band. It proved that the antibody was specificity and had a good performance.6. mortality:In the negative control group, everything was normal, no deaths occurred. In the control group, rats gradually showed a decrease in activity, drowsiness, perioral cyanosis, shortness of breath and some systemic inflammatory response symptoms after LPS injected. It turned grey in the skin and they had shallow breathing when they were moribund.8of12were dead in24h. The mortality was67%.In experimental group, the general condition of rats were obviously better than the control group.2of12were dead in24h. The mortality was17%. The differences among the three groups had statistical significance.7. gross observation of lung tissue:In control group, point-like pulmonary hemorrhage was visible in the lung tissue of the newborn rats after LPS injection, and the degree of bleeding gradually changed from focal to diffuse.Compared with control group, the degree of lung hemorrhage decreased significantly in experimental group.8. pulmonary pathomorphology observation:In the negative control group, the structure of the lung tissue was complete and there was no edema in alveolar septum. In control group, the structure of the lung tissue was incomplete and alveolar septum widened. There were a large number of red blood cells, inflammatory cells and serous exudates in the alveolar. Leukocyte infiltration, capillary hyperemia and expansion was visible in perivascular tissue. Compared with control group, pathological change was significantly reduced in the experimental group.The edema was mild, alveolar septum widened slightly.Also, the leakage of red blood cell and the infiltration of leukocyte reduce significantly.ConclusionsIn this research, we synthesized recombinant plasmid of human thioredoxin protein1at first.It showed that the construction of recombinant plasmid was successful by PCR, enzyme digestion and sequencing detection.Then we transformed the recombinant plasmid into E. coli BL21in order to make prokaryotic expression of human thioredoxin protein1. SDS-PAGE indentification on the expressed protein was performed, which showed the molecular weight as the same as the one that was calculated out by bases number showing prokaryotic expression of human thioredoxin protein1was successful. Using this protein to immune rats for preparing antiserum, the titer could reach1:51200by Elisa detection. Further detection by Western blot, it was proved as a good specificity, and the prepared polyclonal antibody could well bind with human thioredoxin protein1specifically. By this recombinant protein to intervene newborn SD rats before LPS injection, mortality of24hours was significantly decreased, red blood cell leakage and leukocyte infiltration was significantly reduced.It showed that recombinant human thioredoxin protein1had a protective effect to neonatal rats with endotoxemia and laid the foundation for the further experiments. |
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