At present time, the widely used hepatitis B virus( HBV) vaccines consist of only the small hepatitis B surface antigen expressed in yeast or CHO cells. The frequency of non-responders to these vaccines has increased the demand for a more immunogenic vaccine. Some studies have suggested that the addition of preS region to the vaccine will improve its efficacy. However, the large protein (L) containing the whole preS region can not be effectively expressed in vitro. To overcome this problem, two chimeric contructs, SS1, surface gene containing preS1 region at C-terminus and SS2, surface gene containing preS2 region at C-terminus, were constructed and effectively expressed in our previous studies. Here we further constructed an expression vector containing both SS1 and SS2 expression cassettes by separation and ligation the SS2 cassette to a linearized SS1 expression vector pAO815-SS1. The recombinant vector was transformed into Pichia pastoris GS115 by electroporation. A high-level expression strain (GS115-SS1S2) was established by primary screening for His+ transformants and further analysis for induction products. ELISA results demonstrated that the expressed protein had S, preS1 and preS2 antigenicities simultaneously. Western blotting showed that the product can bind to all of the three antibodies, anti-S, anti-preS1 and anti-preS2. The expression protein was present in the form of particles of 20-35 nm diameter and the yield of recombinant particles reached 300-600 mg/L by fermentation. The SS1 and SS2 polypeptides kept intact in purified particles, suggesting that the stability of preS region has been significantly improved.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Epitopes ; genetics ; immunology ; Hepatitis B Surface Antigens ; genetics ; immunology ; Hepatitis B Vaccines ; immunology ; Peptide Fragments ; genetics ; immunology ; Pichia ; genetics ; Protein Precursors ; genetics ; immunology ; Vaccines, Synthetic ; immunology

OBJECTIVETo investigate whether the fetal immune tolerance induction could replace the HLA typing for hematopoietic stem cell transplantation.

METHODSImmune tolerance of SD rats was induced by injecting host Wistar rats peripheral blood mononuclear cells into yolk sac of the embryo, afterward the mature male offsprings were used as donor. The host female recipients received lethal dose irradiation and bone marrow transplantation(BMT). The Wistar rats transplanted with bone marrow from donor and unrelated SD rats as well as the rats which received radiation alone were used as control. The survival, histopathologically GVHD, the mental status, food and water intake, coat characteristics, activities were observed. Forty days after BMT, autologous and allogenous skin transplantation between donor and recipient rats was performed to observe the engraftment of solid organ.

RESULTSThe survival of the rats received bone marrow grafts from the immune tolerant donor was significantly longer than that of control groups (30 day survival rates were 86.7%, 6.7%, 0%, and 0% respectively), and there was no histopathologically GVHD observed, while in the sham group, the manifestations of GVHD was clearly visible. The skin engraftment rate between the host and the immune tolerant donor was significantly higher than that among non-related rats (84.6% and 0% respectively).

CONCLUSIONThe induction of immune tolerance in embryo can overcome the HLA barrier and provide a good donor for hematopoietic stem cell and solid organ transplantation.

Animals ; Embryo, Mammalian ; immunology ; Female ; Graft vs Host Disease ; immunology ; prevention & control ; Hematopoietic Stem Cell Transplantation ; Histocompatibility Testing ; Immunosuppression ; Male ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Transplantation Chimera

The preparation process and immunogenicity of a novel hepatitis B vaccine containing preS1, preS2 and S epitopes were investigated in this study. A Pichia pastoris stain GS115-SS1S2 harbouring two chimeric HBsAg gene constructs, SS1 and SS2 was cultivated by high-density fermentation. 300-600 mg/L of the expression level was achieved through 48-72 h methanol induction. SSIS2 antigen was extracted and purified by silica adsorption, HIC and SEC to 99% purity from the harvested cells. 82 mg purified antigen could be achieved from one liter of fermentation culture. The immunogenicity of the purified antigen was evaluated in NIH mice. Three groups of female NIH mice, 14-16 g in weight, were injected once intraperitoneally with 2.5, 0.625, 0.156 microg of each of the two vaccines: SS1S2 or a commercially available S vaccine. Part of the mice were bled in 30 days after injection to compare the ED50 of the two vaccines. For the SSIS2 vaccine, the ED50 is 0.46, 0.29 and 0.84 microg respectively for the preS1, preS2 and S antigens. For the S vaccine, the ED50 is 0.99 microg for the S antigen. Another part of the mice were bleed in 7 or 14 days to detect preS1, preS2 and S antibodies. Higher ratios of mice were seroconverted for preS1 and preS2 antibodies as compared to the S antibody in these two time points. These results suggest that the SS1S2 vaccine may be more immunogenic than the conventional S vaccines.
Animals ; Epitopes ; immunology ; Female ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B Vaccines ; biosynthesis ; immunology ; Mice ; Pichia ; genetics ; metabolism ; Protein Engineering ; Protein Precursors ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Viral Envelope Proteins ; immunology

Objective To assess the heritability of serum uric acid in adult,using the classic twin design.Methods Adult Twins were recruited from the Qingdao Twin Registry.Uric acid,height,weight were measured.Zygosity in all the same-sex twin pairs was determined by 16 polymorphic markers.Heritability was assessed by structural equation models,with age,gender and body mass index(BMI) included as covariates.Results In total,687 twin pairs were available for data analyses,including 420 pairs of monozygotic and 267 pairs of dizygotic twins.After logarithm transformed,uric acid in males ( 17.47±1.91 ) was significantly higher than in females ( 15.22±1.70,P＜0.0001 ).After adjustment on age,sex and BMI,intraclass correlations for uric acid were 0.70 for monozygotic twins and 0.40 for dizygotic twins.The sex-limitation AE model,combining additive genetic and unique environmental factors,could produce the best fit for the data.Heritability estimate for uric acid was 70.5% (95% CI:65.9-74.6),with the proportion of unique environmental effects as 29.5%(95%CI:25.4-34.2).Conclusion Additive genetic effects appeared to be the major contributor to the variation of uric acid in this twins sample being studied.

The receptor-binding domain(RBD) protein of HCoV-NL63 is a major target in the development of diagnostic assay and vaccine, it has a pivotal role in receptor attachment, viral entry and membrane fusion. In this study, we prepared 2 purified recombinant HCoV-NL63 RBD proteins using in E. coli system and identified the proteins by Western blotting. We first optimized codon and synthesized the RL (232-684aa)coding gene, then amplified the RL or RS(476-616aa) coding gene via PCR using different primers . The RL or RS coding gene was cloned into the pM48 expression vector fused with TrxA tag. The RBD (RL and RS) of HCoV-NL63 were expressed majorly as inclusion body when expressed in E. coli BL21pLys S under different conditions. The expressed products were purified by affinity chromatography then analyzed by SDS-PAGE and Western blotting. Our results showed that the recombinant RBD proteins were maximally expressed at 37 degrees C with 0. 8mM IPTG induction for 4h. RL or RS protein with 95 % purity was obtained and reacted positively with anti-sera from mice immunized with the recombinant vaccinia virus (Tiantan strain) in which HCoV-NL63 RL or RS protein was expressed. In conclusion, the purified recombinant RBD proteins(RL and RS)derived from E. coli were first prepared in China and they might provide a basis for further exploring biological role and vaccine development of HCoV-NL63.
Animals ; Coronavirus Infections ; metabolism ; virology ; Coronavirus NL63, Human ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; Mice ; Mice, Inbred BALB C ; Protein Engineering ; Protein Structure, Tertiary ; Receptors, Virus ; metabolism ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism

OBJECTIVETo establish PEG10 transgenic mice model and study the effect of PEG10 transgene on tumor growth and metastasis in mice.

METHODSThe linearized expression element of pALB-PEG10, which contained mouse albumin promoter, structural gene of PEG10, and polyaenylation signal sequence, was microinjected into 3741 KM mouse fertilized ova. The manipulated embryos were then transplanted into the oviducts of 94 pseudopregnant recipient mice. All the newborn mice were screened by PCR to detect genomic DNA in tail tissue, then PEG10 mRNA and protein expression were detected by RT-PCR and western blot, respectively in the positive mice. Hepatoma cell H22 was subcutaneously inoculated into the right armpit of wild type mice and No.17, No.33 transgenic mice. Tumor size was measured every week. Mice were sacrificed on day 12 and then the tumors were exercised and weighted. Tumors and livers were fixed in formaldehyde and sectioned. The sections were stained with hematoxylin/eosin and examined under microscope. The expression of PEG10 protein was detected with immunohistochemistry method.

RESULTSAmong the 43 off-springs, 3 were positive for tail tissue PEG10 gene examination, PEG10 was successfully expressed in the liver of the randomly selected transgenic mouse. H22 tumor grew faster in all the transgenic mice than in wild type mice. The average size and weight of tumors between the transgenic mice and wild type mice were significantly different (P < 0.05). Most tumors in the transgenic mice invaded the surrounding tissues and showed liver metastasis, PEG10 protein was expressed in liver. In contrast, nearly all the tumors in wild type mice were capsulized and PEG10 was not expressed in liver.

CONCLUSIONOur results showed that the PEG10 gene could be expressed in the liver of the transgenic mice. PEG10 promotes growth, invasion, and metastasis of transplanted H22 tumors in mice.

Animals ; Cell Line, Tumor ; Disease Models, Animal ; Genetic Vectors ; Humans ; Liver ; metabolism ; pathology ; Liver Neoplasms ; metabolism ; pathology ; Mice ; Mice, Transgenic ; genetics ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Neoplasm Transplantation ; Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transgenes ; Xenograft Model Antitumor Assays

Objective　Lipoxin A4 (LXA4) has been proved to have a good protective effect on spinal cord ischemia-reperfusion injury in rats, but whether autophagy is one of the protective mechanisms remains unclear. This study aims to investigate the effects of lipoxin A4 on rat spinal cord ischemia-reperfusion injury.　Methods　48 rats were randomly divided into LXA4 group, ischemia-reperfusion group (SCII group) and sham group with 16 rats in each, and the models of each group were built accordingly. The rats in LXA4 group received intrathecal injection of 10μl LXA4 (300 pmol) 30 minutes after clamping the abdominal aorta. Three groups of rats were sacrificed by cervical dislocation 24 hours after reperfusion and the apoptosis-positive cells were then obtained. The spinal cord tissues of three groups of rats were stained and counted by LC3B fluorescence staining, and the expressions of LC3-II/LC3-I and GABARAP protein were detected by Western blot.　Results　There were few LC3B positive cells in the sham group. Compared to those in the sham group (73.40±19.42), the number of LC3B positive cells in SCII group (399.80±18.46) and LXA4 group (240.80±12.76) significantly increased (P<0.05), and the number in LXA4 group was significantly lower than that in SCII group (P<0.05). The ratio of LC3-II/LC3-I and the expression of GABARAP in SCII group and LXA4 group was significantly higher than those in sham group (P<0.05). The ratio of LC3-II/LC3-I in spinal cord tissue significantly declined compared with that of SCII group (P<0.05).　Conclusion　The autophagy is activated when SCII occurs, indicating that the autophagy is involved in SCII. After LXA4 is administered, autophagy is inhibited and SCII is alleviated.

Anti-tumor traditional Chinese medicine has a long history of clinic application, in which the star molecules have always been the hotspot of modern drug research, but they are limited by the solubility, stability, targeting, bioactivity or toxicity of the monomer components of traditional Chinese medicine anti-tumor star molecules and other pharmacokinetic problems, which hinders the traditional Chinese medicine anti-tumor star molecules for further clinical translation and application. Currently, the nanosystems prepared by supramolecular technologies such as molecular self-assembly and nanomaterial encapsulation have broader application prospects in improving the anti-tumor effect of active components of traditional Chinese medicine, which has attracted extensive attention from scholars at home and abroad. In this paper, we systematically review the research progress in preparation of supramolecular nano-systems from anti-tumor star molecule of traditional Chinese medicine, and summarize the two major categories and ten small classes of carrier-free and carrier-based supramolecular nanosystems and their research cases, and the future development direction is put forward. The purpose of this paper is to provide reference for the research and clinical transformation of using supramolecular technology to improve the clinical application of anti-tumor star molecule of traditional Chinese medicine.

objective To explore the difierence of arousal index between the children with obstructive sleep apnea-hypopnea syndrome(OSAHS)and children with primary snoring.Furthermore,to explore the correlation between the above mentioned arousals and the apnea-hypopnea index(AHI)and lowest oxygen saturation(LSaO2).Methods Between March 2007 and February 2008,102 children suspected of OSAHS underwent overnight polysomnogram monitoring in our medicine sleep center.OSAHS was diagnosed according to the general criterion[Draft of guidelines for the diagnosis and treatment of pediatric sleep apnea hypopnea syndrome(Urumqi)which was published in Chin J Otorhinolaryngol Head Neck Surg in February,2007]. One-hundred and two children were divided into two groups according to the guidelines.Sixty six children[56 boys,10 girls;aged 4-17 years,(7.01±2.24)years(x±s)]who were diagnosed as OSAHS were enrolled in study group and 36 children[29 boys,7 girls;aged 4-13 years,(7.44±2.68)years]who were diagnosed as primary snoring made up control group.The difference of spontaneous arousal index,total arousal index and arousal index related to respiratory stimuli and limb movement were compared between the two groups.Besides this,the correlation between spontaneous arousal index,total arousal index and arousal index related to respiratory stimuli and limb movement and AHI and LSaO2 were also analyzed.Furthermore,the study group were divided into three subgroups according to AHI (≤10 times/h,10 times/h＜AHI≤20 times/h,＞20 times/h).Spontaneous arousal index,total arousal index and arousal index related to respiratory stimuli and limb movement were compared among the three groups.Results The increased total aronsal index and arousal index related to respiratory stimuli and the decreased spontaneous arousal index of study group were significant as compared to those of control group (Mann-Whitney U,z value,-3.148,-3.866,-2.791;P value,0.002,0.000,0.005,respectively).The increased arousal index related to respiratory stimuli were significant as being compared among the three groups.Arousal index related to respiratory stimuli wag correlated with AHI(COefficient correlation:0.734.P=0.000).Other kinds of arousals and arousal index related to respiratory stimuli were not correlated with LSaO2(Spearman rank correlation analysis).When compared to control group,stage I increased and stage REM decreased and the difference was significant(z were-2.423,-3.519;P were 0.015,0.000).Conclusions The arousal index related to respiratory stimuli were increased and spontaneous arousal index were decreased in children with OSAHS.Arousal index related to respiratory stimuli is more suitable to show the degree of sleep fragment than other arousal index.

Objectives: An Asian Gynecologic Oncology Group phase III randomized trial was conducted to determine whether maintenance chemotherapy could improve progression-free survival (PFS) in stages III/IV ovarian cancer. Methods: Between 2007 and 2014, 45 newly-diagnosed ovarian cancer patients were enrolled after complete remission and randomized (1:1) to arm A (4-weekly carboplatin area under the curve 4 and pegylated liposomal doxorubicin [PLD] 30 mg/m2, n=24) for 6 cycles or arm B (observation, n=21). The primary end-point was PFS. A post hoc translational study was conducted to deep sequence BRCA/homologous recombination deficiency (HRD) genes, because BRCA/HRD mutations (BRCA/HRDm) are known to be associated with better prognosis. Results: Enrollment was slow, accrual was closed when 7+ years had passed. With a medianfollow-up of 88.9 months, the median PFS was significantly better in arm A (55.5 months) than arm B (9.2 months) (hazard ratio [HR]=0.40; 95% confidence interval [CI]=0.19–0.87; p=0.020), yet the median overall survival was not significantly different in arm A (not reached) than arm B (95.1 months) (p=0.148). Overall grade 3/4 adverse events were more frequent in arm A than arm B (60.9% vs 0.0%) (p<0.001). Quality of life was generally not significantly different. Distribution of BRCA1/2m or BRCA/HRDm was not significantly biased between the two arms. Wild-type BRCAon-HRD subgroup seemed to fare better with maintenance therapy (HR=0.35; 95% CI=0.11–1.18; p=0.091). Conclusions Despite limitations in small sample size, it suggests that maintenance carboplatin-PLD chemotherapy could improve PFS in advanced ovarian cancer.