Construction and eukaryotic expression of recombinant HSA-TP5 fusion gene expression vector / 吉林大学学报(医学版)

Objective:To construct the expression vector of the fusion protein of human serum albumin (HSA) and thymopentin (TP5) and to express it in Pichia pastoris,and to elucidate the biological activity of fusion protein.Methods:The HSA-TP5 fusion gene was constructed by gene recombination and transfected into Pichia pastoris to construct the eukaryotic expression system of HSA-TP5.The recombinant eukaryotic expression plasmid of PPICZα-HSA-TP5 was obtained by agarose gel electrophoresis and purification reagent.The two step fermentation method was used to ferment gene engineering bacteria of HSA-TP5 in high density,and the fermentation supernatant protein was precipitated and concentrated;the purified fusion protein was obtained by cation exchange chromatography and hydrophobic chromatography and analyzed by SDS-polyacrylamide gel electrophoresis.The effect of the fusion protein on the proliferation of lymphocytes was detected by MTT assay.Results:The HSA target gene fragment with length of 1 845 bp was achieved by PCR method.The HSA-TP5-pPICZαC fusion plasmid was identified by restriction endonuclease digestion,and the fragment length was 707 bp.The sequence analysis showed that the HSA and TP5 sequences of the target genes were identical with the gene sequences reported in GenBank and were fused by forward fusion.PCR method confirmed that the eukaryotic recombinant plasmid PPICZ αC-HSA-TP5 was integrated into the yeast genome,and compared with control group,the target gene PCR product length was found to be 1 860 bp.SDS-PAGE analysis showed that the expression level of HSA-TP5 fusion protein was gradually increased with the induction time within 72 h.HSA-TP5 fusion protein was purified by cation exchange chromatography and AKTA multifunctional protein purification system.The MTT assay results showed that HSA-TP5 fusion protein was consistent with TP5 protein in promoting lymphocyte proliferation activity.Conclusion:HSA-TP5 fusion protein can be obtained by constructing the eukaryotic expression system of Pichia pastoris and owns the biological activity.

Construction and eukaryotic expression of recombinant HSA-TP5 fusion gene expression vector / 吉林大学学报(医学版)

Objective:To construct the expression vector of the fusion protein of human serum albumin (HSA) and thymopentin (TP5) and to express it in Pichia pastoris,and to elucidate the biological activity of fusion protein.Methods:The HSA-TP5 fusion gene was constructed by gene recombination and transfected into Pichia pastoris to construct the eukaryotic expression system of HSA-TP5.The recombinant eukaryotic expression plasmid of PPICZα-HSA-TP5 was obtained by agarose gel electrophoresis and purification reagent.The two step fermentation method was used to ferment gene engineering bacteria of HSA-TP5 in high density,and the fermentation supernatant protein was precipitated and concentrated;the purified fusion protein was obtained by cation exchange chromatography and hydrophobic chromatography and analyzed by SDS-polyacrylamide gel electrophoresis.The effect of the fusion protein on the proliferation of lymphocytes was detected by MTT assay.Results:The HSA target gene fragment with length of 1 845 bp was achieved by PCR method.The HSA-TP5-pPICZαC fusion plasmid was identified by restriction endonuclease digestion,and the fragment length was 707 bp.The sequence analysis showed that the HSA and TP5 sequences of the target genes were identical with the gene sequences reported in GenBank and were fused by forward fusion.PCR method confirmed that the eukaryotic recombinant plasmid PPICZ αC-HSA-TP5 was integrated into the yeast genome,and compared with control group,the target gene PCR product length was found to be 1 860 bp.SDS-PAGE analysis showed that the expression level of HSA-TP5 fusion protein was gradually increased with the induction time within 72 h.HSA-TP5 fusion protein was purified by cation exchange chromatography and AKTA multifunctional protein purification system.The MTT assay results showed that HSA-TP5 fusion protein was consistent with TP5 protein in promoting lymphocyte proliferation activity.Conclusion:HSA-TP5 fusion protein can be obtained by constructing the eukaryotic expression system of Pichia pastoris and owns the biological activity.

High yields and soluble expression of superoxide dismutases in Escherichia coli due to the HIV-1 Tat peptide via increases in mRNA transcription

This study aimed to validate the high yield and soluble expression of proteins carrying the transactivator of transcription (Tat) peptide tag, and further explored the potential mechanism by which the Tat tag increases expression. Escherichia coli superoxide dismutase (SOD) proteins, including SodA, SodB and SodC, were selected for analysis. As expected, the yields and the solubility of Tat-tagged proteins were higher than those of Tat-free proteins, and similar results were observed for the total SOD enzyme activity. Bacterial cells that overexpressed Tat-tagged proteins exhibited increased anti-paraquat activity compared with those expressing Tat-free proteins that manifested as SodA>SodC>SodB. When compared with an MG1655 wild-type strain, the growth of a ΔSodA mutant strain was found to be inhibited after paraquat treatment; the growth of ΔSodB and ΔSodC mutant strains was also slightly inhibited. The mRNA transcript level of genes encoding Tat-tagged proteins was higher than that of genes encoding Tat-free proteins. Furthermore, the α-helix and turn of Tat-tagged proteins were higher than those of Tat-free proteins, but the β-sheet and random coil content was lower. These results indicated that the incorporation of the Tat core peptide as a significant basic membrane transduction peptide in fusion proteins could increase mRNA transcripts and promote the high yield and soluble expression of heterologous proteins in E. coli.

Severe neonatal hypoxic-ischemic encephalopathy: clinical features and follow-up of 123 cases / 中国新生儿科杂志

Objective To study the clinical features and follow-up of newborns with severe hypoxic-ischemic encephalopathy ( HIE) , and to provide the basis for rational diagnosis, treatment and follow-up.Methods Clinical data of cases of HIE from the Neonatal Department of our Hospital from January 2011 to October 2014 were studied retrospectively. The data of general information, laboratory examination, treatment, outcome, follow-up and prognosis of the patients were collected. Multivariate logistic regression analysis was used to study the influential factors of the prognosis of HIE.Results A total of 123 infants with sever HIE were enrolled in our study. In addition to general therapy, 6 cases were treated with mild hypothermia, and 21 cases were treated with high pressure oxygen. 60 cases improved our treatment, 55 cases had withdrawal treatment with parental consent, and 8 cases died. Single factor analysis showed that 5 minutes Apgar score, convulsions, coma, pH, BE, organ injury, and mild hypothermia treatment were the risk factors that affect the prognosis of severe HIE. Multiple factors analysis showed that 5 min Apgar score <3 points ( OR=4. 071 ,95℅CI 1. 309-15. 613 ) and BE≤-10 mmol/L ( OR=36. 810, 95℅CI 5. 913-41. 119) were independent risk factors of prognosis of severe HIE ( P<0. 05). Hospitalization within the first 72 hours of life ( OR=0. 096, 95℅CI 0. 096-0. 353) was a protective factor of severe HIE. Multiorgan injury ( mainly the injury of brain, lung and heart) and electrolyte imbalance ( mainly hypocalcemia and hyponatremia ) were common complications of serve HIE. In the follow-up of these patients, 33 cases were loss in follow up, and 49 cases died (8 cases died during hospitalization, 41 cases died after withdrawal of treatment). The top five causes of death were abandonment of treatment due to financial reasons and the fear of adverse outcome (n=20), multiple organ dysfunction ( n =16 ) , and pneumothorax ( n =4 ) , diffuse intravascular coagulation (n=6), and shock (n=3). 41 cases survived were followed up for 9~54 months. The critical clinical conditions observed among these infants included cerebral palsy ( n = 5 ) , epilepsy ( n = 3 ) and developmental retardation(n=26).Conclusions There are many complications of severe HIE.The mortality of severe HIE is high, and the incidence of poor outcome of survivors is also high. Timely detection of risk factors is the key to the prevention of severe HIE. Long-term prognosis of severe HIE requires proper organization of neonatal follow up.

Construction of secretory expression vector of rhKD/APPvar and expression and purification of its recombinant protein in Pichia pastoris / 吉林大学学报(医学版)

Objective To construct the engineering bacteria expressing the recombinant human Kunitz protease inhibitor domain of amyloid protein precursor variant (rhKD/APPvar)in Pichia pastoris,and to establish the methods suitable for large-scale fermentation and purification of rhKD/APPvar.Methods The rhKD/APPvar expression vector was constructed based on the rhKD/APPvar-pPICZαexpression vector. Two restriction enzyme loci (ApaⅠ and SacⅡ)were added to two flanks of KD/APP and human KD/APP activity center RAM was replaced by the active site of BPTI KAR.After the rhKD/APPvar-pPICZαexpression vector was transformed into Pichiapastoris,optimized expression and purification of rhKD/APPvar was performed.The rhKD/APPvar was purified with cation exchange chromatography and desalting.Results The results of digestion identification and DNA sequencing analysis demonstrated that the recombinant plasmid rhKD/APPvar-pPICZα was successfully constructed and transfected into pastoris X-33. The SDS-PAGE analysis results indicated that rhKD/APPvar expressed after the induction of methanol and the relative molecular weight was 6 700.After a series of experiments the optimal expression conditions of rhKD/APPvar were obtained as follows:the optimal pH was 6.0 and the optimal induction time point was about the 5 th day for the strain.After purified the purity of rhKD/APPvar was about 95%.Conclusion KD/APPvar-pPICZ is successfully constructed;after expression in Pichia pastoris and purification,the rhKD/APPvar protein is obtained.

ACTH, cortisol and aldosterone level of preterm infants with critical illness / 中国小儿急救医学

Objective To investigate the effect of illness severity on preterm infant's hypothalamusputituary-adrenal (HPA) axis, we measured the serum concentration of cortisol,aldosterone and adrenocorticotropic hormone (ACTH). Methods Ninety preterm infants who were transferred to our hospital within 72 hours after birth were involved. These preterm infants were divided into two groups:gestational age (GA) ≥34 weeks' preterm infants and GA <34 weeks' preterm infants. We evaluated these preterm infants at the time of admission,day 7 and day 14 after birth with neonatal critical illness score (NCIS). Then they were divided into mild group and severe group by the lowest score. We measured their serum cortisol,aldosterone and ACTH at the time of admission,day 7 and day l4 after birth. Results (1) The serum cortisol concentration of preterm infants with severe illness was higher than that of preterm infants with mild illness. Among the GA ≥34 weeks' preterm infants,the serum cortisol concentration of preterm infants with severe illness was significandy higher than that of preterm infants with mild illness within 72 hours after birth (t = -2.263,P =0. 029). Among the GA <34 weeks' preterm infants,the serum cortisol concentration of preterm infants with severe illness was significantly higher than that of preterm infants with mild illness on day 14 after birth (t =-2. 913 ,P =0. 006). (2) Among the preterm infants with severe illness,the serum cortisol concentration of the GA≥34 weeks' was significantly higher than that of the GA < 34 weeks' within 72 hours after birth (t =-2. 641 ,P =0. 010) ;the serum cortisol concentration of the GA <34 weeks' was significantly higher than that of the GA≥34 weeks' on the day 14 after birth(t = -2. 189,P =0. 036) . (3) The serum cortisol concentration was significantly decreased in the GA≥34 weeks'preterm infants (F = 4. 679, P =0. 012). (4) The serum cortisol concentration of aldosterone and ACTH was not significantly different between preterm infants with severe illness and those with mild illness. Conclusion The preterm infant already has the ability to respond to stimuli by regulating cortisol secretion. The serum cortisol concentration increases as disease severity worsens.Serum aldosterone and ACTH concentration are not correlated with the severity of the disease.

Reconstruction of Tissue Engineering Skin by Epidermal Cells and Fibroblasts Combined with Modified Polymer of Lactic Acid / 现代生物医学进展

Objective: To investigate the applied feasibility of scaffold with modified PLA (Polymer of lactic acid) in tissue engineering. Methods:First, we adopted salting-in method to prepare porous foam scaffold. Then, we reconstructed tissue engineering skin by epidermal cells and fibroblasts combined with modified PLA. On the 14th day of cell culturing in vitro, we was a control. Results:The arfificial skin is composed of epidermis and dermis and similar to natural skin in appearance. The skin consists of fibroblasts and keratinocytes, which are in various proliferation and differentiation stages. Fibroblasts and keratinocytes distribute on the surface of polymer of lactic acid (PLA) and the number of fibroblast and keratinocyte increase. Conclusion:Dialdehyde starches (DAS) not only improve the function of PLA but also have good effects on cells. Moreover, it does not affect the growth and the metabolism of the cells. So it is feasible to use modified scaffold to construct tissue engineering skin.

Animal grafting experiment of reconstructive tissue engineered artificial skin made by chitosan as stromal scaffold / 中国组织工程研究

BACKGROUND: Skin transplantation is the most effective conventional method to cure large area full-thickness skin damage caused by empyrosis or some diseases, but present deficiency of skin donator is the largest barrier in front. The most ideal way to solve this problem is to use tissue-engineering skin reconstructed by self-skin cells as seed cells.OBJECTIVE: To investigate the effects of tissue engineered artificial animal skin in animal grafting experiment.DESIGN: A randomized controlled trial SETTING: Institute of frontier medical sciences and department of dermatology in a university.MATERIALS: Study was performed in the Cell-Engineering Institute of Jilin University from September 1998 to July 2001. Totally 20 newborn Wistar rats and 24 8-week old male nude mice were selected into our study.METHODS: Full-thickness wounds(diameter: 20 nn) were made on the backs of twenty-four nude mice to establish full-thickness skin defect animal model for grafting by the tissue-engineered reconstructive artificial skin made by chitosan(CH) as stromal scaffold. Twenty-four 8-week old nude mice were divided into artificial skin (AS) group, chitosan membrane(CH) group and control group (CG). All wounds were covered with AS, CH or petrolatun gauze correspondingly. The wounds of each groups were observed daily,techniques like histology and infrared-ray scan were used for a dynamical surveillance on the 3rd, 7th, 14th and 21st days.MAIN OUTCOME MEASURES: ① general observation; ② blood supply in recipient area under infrared-ray observation; ③ histological observation.RESULTS: Transplanted AS had a favorable fusion between tissue-engineered skin and self-skin on the 3rd day after grafting with a few of ingrowths of capillaries. The color of the AS was closed to self-skin. The capillaries gradually increased in the grafts over time. The new epidermis was clearly consisted of stratum basale, stratum spinosum, stratum granulosum, and stratum corneum. Keratinization enhanced with exfoliation. Cells in dermis increased and the scaffold gradually degraded. The secreted extracellular matrix increased as well. On the 14th day after grafting, the wounds almost recovered.The color of the grafted artificial skin was more similar to the nature skin with very little scaring, which indicated that a second grafting was unnecessary. The scabs did not completely fall off in CH group until the 14th day, and the wound was not healed. The color was darker than that of AS group. The scabs fell off in CG, and the wounds were big and deep with dark red color.CONCLUSION: The new reconstructive tissue-engineered artificial skin with CH as stromal scaffold has good histocompatibility, which can be applied in grafting for full-thickness wounds.

Separation and long-term cultivation of rat hepatocytes / 吉林大学学报(医学版)

Objective :To study a simplified method of isolation of rat hepatocytes and to observe the pro-cess of cell morphology in long-term culture. Methods :Rat hepatocytes were isolated by a single two-stepperfusion method. The yield and viability were assessed by trypan blue exclusion. [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide] (MTT) was used to test the effect of serum concentration of newborn calf serum on the proliferation of hepatocytes. Hepatocytes were inoculated in the culture mediumconsisted of Williams' E supplemented with insulin,dexamethasone and 10％ new born calf serum. Themorphologic change of cultured hepatocytes was observed. Results:The average yield of hepatocytes was 2.26× 108 cells per rat, with an average viability of 95.6％. New born calf serum had strong biological activi-ty to stimulate the proliferation of hepatocytes and there was close-effect relationship followed by the in-crease of new born calf serum concentration. The rat hepatocytes can be cultured for 5～ 6 weeks withpreservation of normal morphologic appearance. Conclusion:The rat hepatocytes isolated by the abovemethod have high yields and viability and can be long-term cultured in vitro.

The inhibitory action of rhTRAIL on mouse breast carcinoma / 中国病理生理杂志

AIM:To explore the inhibitory action of recombinant human tumor necrosis factor-related apoptosis-inducing ligand(rhTRAIL) on mouse breast cancer. METHODS:Each mouse was inoculated 0.2 mL (1?106) D2F2 cells subcutaneously in the right lower limb and they were divided into five groups randomly. The control group was infused PBS 0.2 mL,while the low-dose,medium,high groups received purified rhTRAIL 2.5 mg/kg,5.0 mg/kg,10.0 mg/kg,respectively,the positive group was administered cyclophosphamide 30.0 mg/kg. Every group was operated by peritoneal injection once a day for fifteen days. The mice were weighed every day. The growth state was viewed and the size of the tumor was measured every 3 d to calculate the tumor volume and tumor suppression rate. All mice were killed after 15 d. The pathologic changes of the tumor were observed under light-microscopy and electronic microscopy. The cell cycle and apoptosis index of D2F2 cells were analyzed by flow cytometry. RESULTS:The body weight and tumor volume in low-dose,medium,high groups were lower than those in control group and the restriction effect was more significant than that in the control group (P