Drug resistance and molecular typing of diarrheagenic escherichia coli from the diarrhea clinics in Qingpu District, Shanghai / 中华预防医学杂志

Objective: To analyze the drug resistance and multilocus sequence typing of five types of diarrheagenic Escherichia coli (DEC) isolated from diarrhea outpatients of diarrhea comprehensive monitoring designated hospital in Qingpu District, Shanghai from 2015 to 2019. Methods: From January 2015 to December 2019, five types of DEC, isolated and identified from diarrhea outpatient cases' anal swabs of the Qingpu branch of Zhongshan Hospital were collected to determine the minimal inhibitory concentration by using the micro broth dilution susceptibility test. The strains, resistant to the third-generation cephalosporins or carbapenems, or producing ESBLs, were selected based on the results of sensitivity tests and determined by WGS. The MLST typing of DEC was analyzed based on the WGS technology and the minimum spanning tree was constructed by BioNumerics 7.6 software to analyze the local dominant flora. Results: A total of 513 strains of DEC were detected and isolated from 4 494 anal swabs, with a detection rate of 11.42%. About 500 strains were tested for drug sensitivity to nine antibiotics in four classes, including 330 strains of enterotoxigenic E.coli (ETEC), 72 strains of enteroaggregative E.coli (EAEC), 95 strains of enteropathogenic E.coli (EPEC), 1 strain of enterohemorrhagic E.coli (EHEC), and 2 strains of enteroinvasive E.coli (EIEC). From 2015 to 2019, the resistance rate of cefotaxime-clavulanic acid was significantly different (P<0.05). The resistance rate of virulence types of DEC to nalixic acid was significantly different (P<0.05). About 71 strains of DEC were determined by WGS, and 77 drug-resistant genes were detected. Strains were classified into 32 ST subtypes, with the dominant genotypes being ST-1491 (29.6%, 21/71) and ST-10 Complex (23.9%, 17/71). All ST-1491 produced ESBLs, which were blaCTX-M gene mutant strains. The dominant type of ST-10 complex was ST-218 (35.3%, 6/17). In addition, 8 strains of EAEC, 14 strains of EPEC and 49 strains of ETEC were classified into 7, 14 and 18 ST subtypes, respectively. Conclusion: The drug resistance of DEC strains from the diarrhea outpatient case of Qingpu District is serious. The ST types of EAEC and EPEC are highly polymorphic. The dominant ST types of DEC are basically consistent with the common genotypes in southeast China.

Protective Effect of Xiangsha Yuyang Decoction on Oxidative Injury in Rats with Gastric Ulcer Based on p62/Keap1/Nrf2 Signal Pathway / 中国实验方剂学杂志

Objective:To confirm the protective effect of Xiangsha Yuyang decoction on acetic acid-induced gastric ulcer model rats and explore its mechanism， so as to provide experimental basis for clinical drug use. Method:The 60 SPF Wistar rats were randomly divided into 6 groups： group， model group， high， middle and low dose groups of Xiangsha Yuyang decoction and omeprazole control group. The rat model of gastric ulcer was induced by acetic acid. The rats in the high， middle and low dose groups of Xiangsha Yuyang decoction were intragastrically administered at the dose of 28，14，7 g·kg^-1， and with omeprazole at the dose of 4.17 g·kg^-1in normal saline， respectively. The rats in the blank group and model group were intragastrically infused with the same volume of normal saline once a day. After 14 days of continuous treatment， the rats were killed， the blood was collected， the area and inhibition rate of gastric ulcer were measured and calculated， the histopathological sections of gastric mucosa were made and the state of gastric mucosal injury was observed， and the changes of gastric mucosal repair factor， gastric tissue related protein， oxidative stress factor and inflammatory factor in serum were detected by enzyme-linked immunosorbent assay（ELISA）. Detected the expression of p62 Kelch-like epichlorohydrin-related protein 1 （Keap1）， nuclear transcription factor E2 related factor 2 （Nrf2） and heme oxygenase-1 （HO-1） signal pathway-related proteins in gastric mucosa by Western blot. Result:Compared with control group， the gastric mucosa of the model group showed obvious pathological changes and a large number of leukocytes infiltrated. In model group， the ulcer area was significantly increased（<italic>P</italic><0.01）， the contents of mucin mucoprotein 5AC （MUC5AC）， epidermal growth factor （EGF）， superoxide dismutase （SOD） and increased prostaglandin E₂ （PGE₂） were significantly decreased（<italic>P</italic><0.01）， the gastrin （GAS）， 8-hydroxydeoxyguanosine （8-OHdG）， malondialdehyde （MDA）， tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>）， cyclooxygenase-2 （COX-2） were significantly increased. The expression of HO-1 and Nrf2 protein decreased significantly（<italic>P</italic><0.01）， the content of Keap1 increased significantly （<italic>P</italic><0.05）， and the expression of p62 protein decreased. Compared with model group， the hierarchical structure of cells in Xiangsha Yuyang decoction high dose group and omeprazole group were clearer and regular， middle and low dose groups could also repair gastric mucosa to a certain extent. The high and middle dose groups of Xiangsha Yuyang decoction could significantly reduce the gastric ulcer area of acetic acid-induced gastric ulcer rat model （<italic>P</italic><0.01） and increase the ulcer inhibition rate. It can effectively promote the expression of MUC5AC and EGF in gastric mucosa， decrease the level of GAS（<italic>P</italic><0.05，<italic>P</italic><0.01）， decrease the level of 8-OHdG and MDA， increase the activity of SOD（<italic>P</italic><0.01）， decrease the expression level of TNF-<italic>α</italic> and COX-2， increase the content of PGE₂， and significantly increase the amount of Nrf2 and HO-1 protein in gastric mucosa（<italic>P</italic><0.01）. The high dose group of Xiangsha Yuyang decoction could decrease the protein expression of Keap1（<italic>P</italic><0.05） and increase the expression of p62 protein. Conclusion:Xiangsha Yuyang decoction is effective in the treatment of acetic acid-induced gastric ulcer model rats， which can effectively reduce the ulcer area， increase the ulcer inhibition rate and protect the ulcer tissue. Its mechanism may be related to activating p62/Keap1/Nrf2 signal pathway and regulating the expression of related genes so as to improve inflammatory response and regulate oxidative stress response.

Lignans from seed of Hydnocarpus anthelminthica / 中国中药杂志

This project is to investigate lignans from the seed of Hydnocarpus anthelminthica. Thirteen lignans were isolated from the 95% ethanol extract of the seed of H. anthelminthica, by polyamide resin, Sephadex LH-20, ODS column chromatography and preparative HPLC. Their structures were elucidated as(+)-syringaresinol(1), lirioresinol A(2),(+)-medioresinol(3),(7R,8R,8'R)-4'-guaiacylglyceryl-evofolin B(4), leptolepisol C(5),(-)-(7R,7'R,7″R,8S,8'S,8″S)-4',4″-dihydroxy-3,3',3″,5,5',5″-hexamethoxy-7,9':7',9-diepoxy-4,8″-oxy-8,8'-sesquineolignan-7″,9″-diol(6),(-)-(7R,7'R,7″R,8S,8'S,8″S)-4',4″-dihydroxy-3,3',3″,5,5'-pentamethoxy-7,9':7',9-diepoxy-4,8″-oxy-8,8'ses-quineolignan-7″,9″-diol(7), ceplignan(8), hydnocarpusol(9), isohydnocarpin(10),(-)-hydnocarpin(11), hydnocarpin(12), and hydnocarpin-D(13) by spectroscopic data analysis. Compounds 1-8 were obtained from the genus Hydnocarpus for the first time.

Chemical compositions from roots of Erythrina corallodendron / 中国中药杂志

This project is to investigate chemical compositions from the roots of Erythrina corallodendron. Through the methods of silica gel,ODS,Sephadex LH-20 column chromatography and preparative HPLC,15 compounds were isolated from the 95% ethanol extract of the roots of E. corallodendron. Based on spectroscopic techniques,the structures of these compounds were identified as 10,11-dioxoerythraline( 1),erythrinine( 2),erythraline( 3),11-methoxyerythraline( 4),cristanines B( 5),erythratine( 6),erysotrine( 7),medioresinol( 8),( ±)-ficusesquilignan A( 9),( +)-pinoresinol( 10),nicotinic acid( 11),dibutyl phthalate( 12),vanillic acid( 13),3-hydroxy-1-( 4-hydroxy-3-methoxyphenyl)-1-propanone( 14),and syringic acid( 15). Compounds 8-10 are isolated from genus Erythrina for the first time and all compounds are isolated from E. corallodendron for the first time. Furthermore,this paper screened the antioxidant and cytotoxic activities of the compounds using models of liver microsomal oxidation inhibition and MTT.

The protective effects of leonurine against acute endotoxin induced uveitis in rats / 复旦学报（医学版）

Objective To investigate whether the protective effects of leonurine (SCM-198) against endotoxin induced uveitis (EIU) of SD rats caused by lipopolysaccharide (LPS) was existing,and discuss the underlying mechanisms.Methods Thirty-six normal healthy male SD rats were divided into 3 groups randomly with the same baseline bodyweight and feeding conditions.All rats received intragastric administration every day.The experimental group was devided into 4 subgroups,rats in these subgroups received SCM-198 intragastric administration by as the dose of 10,20,40 and 80 mg/kg bodyweight per day,rats in the negative control group received intragastric administration of normal saline 10 mL/kg per day,rats in the positive control group received intragastric administration ofdexamethasone (DEX) 0.5 mg/kg bodyweight per day.All rats received a 21-day-intragastric administration.The body weight of all rats was monitored every 7 days.The electroretinogram (ERG) examination was taken in the 18th day.All rats received a 100 mg S.typhi LPS intraperitoneal injection after the 21st intragastric administration.Twenty-four hours later,following anaesthesia,all rats received another ERG examination,and inflammation was scoring under microscope by 2 experienced ophthalmologists,after that the aqueous humor of all rats was collected from the left eye.The aqueous humor was kept in-80 ℃ immediately.Then the rats were sacrificed and the right eyes were immediately enucleated to finish the HE staining and immunohistochemistry (IHC) staining examination of tumor necrosis factor-α (TNF-α) and intercellular cell adhesion molecule-1 (ICAM-1).The total amount of protein in aqueous humor was detected by BCA test.Western blot was used to examine the expression of TNF-α,interleukin-1β (IL-1β),IL-6 and ICAM-1.All data was analyzed by SPSS 19.0,and differences were considered significant at P＜0.05.Results The body weight of the rats in positive control group was significantly lower (P＜0.05) than the experimental group and the negative control group after the 21-day-intragastric administration.The inflammatory score of experimental group was lower than that of the negative control group,but higher than the score of positive control group.The HE staining sections showed the similar results.The a wave of ERG in 0.01 cd of rats received 20 mg/kg SCM-198 daily intragastric administration after LPS injection was significantly lower than that before the LPS injection (P＜0.05),also lower than other groups after LPS injection.The expression of TNF-α,IL-6 and IL-1β in the aqueous humor of the rats in the subgroup of SCM-198 10 mg/kg daily intragastric administration was lower than other groups.Conclusions Intragastric administration of SCM-198 has protective effect against endotoxin induced uveitis in SD rats without obvious adverse reaction,which could alleviate the imflammatory reaction and the damage to the uvea construction.NF-κB plays an important role in the reaction.Thus,SCM-198 is a candidate potent compound with potential therapeutic applications in inflammation associated eye diseases.While the best mode and dose of administration should be further investigated.

Efficacies of intrauterine balloon tamponade, intrauterine gauze tamponade and B-Lynch suture in prevention and treatment of postpartum hemorrhage / 第二军医大学学报

Objective To investigate the efficacies of intrauterine balloon tamponade, intrauterine gauze tamponade and B-Lynch suture in prevention and treatment of postpartum hemorrhage. Methods A total of 266 patients with high risk of postpartum hemorrhage or postpartum hemorrhage in our hospital from January 2013 to October 2014 were included in this study. They received intrauterine Bakri balloon tamponade (n=114), intrauterine gauze tamponade (n=69) or B-Lynch suture (n=83). The hemostatic effects of three methods and their relationship with different hemostasis occasions and high risk factors were analyzed. Results The hemostatic rates of intrauterine balloon tamponade, gauze tamponade and B-Lynch suture were 90.4%, 94.2% and 92.8%, respectively, with no significant differences found between the 3 groups (P>0.05). The hospitalization time, postpartum infection or involution of the uterus of three methods were not significantly different (P>0.05). The operation period was (46.08±13.8) min for intrauterine Bakri balloon tamponade, (56.49±12.94) min for intrauterine gauze tamponade, and (52.36±21.11) min for B-Lynch suture,with that of Bakri balloon group being significantly shorter than the other two groups (P<0.01). As for hemostatic occasion, 134 cases received preventive hemostatic treatment and were all successful, while 132 cases receiving hemostatic treatment had a successful rate of 84.1%, being significantly lower than that of the prevention hemostasis group (P<0.01); moreover, the postpartum infection rate was significantly lower in the prevention group compared with hemostatic treatment group (P<0.01). When placenta factor and uterine factor exist at the same time, the bleeding rate and bleeding volume of the mixed factor were significantly higher than that of single factor (P<0.01). Conclusion The three hemostasis methods have no differences in their hemostatic effects, hospitalization time, postpartum infection and uterine involution, with Bakri balloon's operation having the shortest time, therefore it may serve as an emergency hemostasis method to win more time for further treatment. The success rate of hemostasis depends not on the hemostasis method, but on the right occasions, the earlier the better. When placenta factor and uterine factor exist at the same time, the failure rate of hemostasis is higher, and emergency plans should be taken to reduce postpartum hemorrhage as soon as possible.

Expressions of Mast Cell Tryptase and Brain Natriuretic Peptide in Myocardium of Sudden Death due to Hypersensitivity and Coronary Atherosclerotic Heart Disease / 法医学杂志

OBJECTIVES@#To explore the value of mast cell tryptase and brain natriuretic peptide（BNP） in the differential diagnostic of sudden death due to hypersensitivity and coronary atherosclerotic heart disease.@*METHODS@#Totally 30 myocardial samples were collected from the autopsy cases in the Department of Forensic Pathology, Shanxi Medical University during 2010-2015. All samples were divided into three groups： death of craniocerebral injury group, sudden death of hypersensitivity group and sudden death of coronary atherosclerotic heart disease group, 10 cases in each group. Mast cell tryptase and BNP in myocardium were detected by immunofluorescence staining and Western Blotting.@*RESULTS@#Immunofluorescence staining showed that the positive staining mast cell tryptase appeared in myocardium of sudden death of hypersensitivity group and coronary atherosclerotic heart disease group. Among the three groups, the expression of mast cell tryptase showed significantly differences through pairwise comparison （P<0.05）; The expression level of BNP in sudden death of coronary atherosclerotic heart disease group were significantly higher than the sudden death of hypersensitivity group and death of craniocerebral injury group （P<0.05）. The difference of the expression level of BNP between the sudden death of hypersensitivity group and the death of craniocerebral injury group had no statistical significance （P>0.05）.@*CONCLUSIONS@#The combined detection of the mast cell tryptase and BNP in myocardium is expected to provide help for the forensic differential diagnosis of sudden death due to hypersensitivity and coronary atherosclerotic heart disease.

Personality characteristics with fifteen factors questionnaire plus of pharmacists / 中华行为医学与脑科学杂志

Objective To explore the personality traits and professional profile of pharmacists, and compare the personality with other majors. Methods This study used 15FQ + personality factor questionnaire for comparing 92 pharmacists,who had been working more than 5 years in pharmacy department of general hospital,with 1064 other different professional staffs,who also had been working more than 5 years. Results The results showed that fA (17.61 ±3.58),fG (16.05 ±5.00),fI (15.82 ±4.26) ,tN (16.74 ±3.78) and fQ3 (18.04 ±3.28)scores of pharmacists were significant higher (P ＜ 0. 05 ), while fC ( 10.61 ± 4.16 ), fE ( 10.84 ± 4.14 ), fL ( 6.33± 4.15 ) and fQ2 ( 8.37 ± 3.94) scores were significant lower (P ＜ 0.05 ). Comparing pharmacists with dentists,fL was significant higher (P＜ 0. 05 ). Comparing with clinical and medical imaging profession, there were significant difference on fA,fI,fL, fM, fQ2 (P ＜ 0.05 ). Comparing with liberal arts and engineering, there were significant difference on fB, fC, fE, fI, fL, fQ1, fQ2 (P ＜ 0. 05 ). Conclusion Comparing with other different professional staffs ,pharmacists have their own personality traits, which are positive, perseverance, sensitive serious, self-discipline and so on. It provides a scientific basis for pharmacists selection and training.

A new study on diffusion tensor imaging of the whole visual pathway fiber bundle and clinical application / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>With conventional imaging methods only the morphous of the visual nerve fiber bundles can be demonstrated, while the earlier period functional changes can not be demonstrated. We hypothesized that diffusion tensor imaging (DTI) would demonstrated the whole optic never fiber bundle and visual pathway and the earlier period functional changes. The purpose of the present study was to evaluate the application of DTI technique in the demonstration of the whole optic never fiber bundle and visual pathway, and the influence of orbital tumors on them.</p><p><b>METHODS</b>GE 1.5 T signa HD MR System, and the software package DTV2 were adopted. The total 45 subjects were enrolled, including 15 volunteers and 30 patients. All patients had ocular proptosis from minor to major. Seven patients had visual acuity decrescence.</p><p><b>RESULTS</b>The nerve fiber bundles, e.g. optic chiasma, optic tract and optic radiation in posterior visual pathway were well demonstrated in all cases. Wherein, the intact whole visual pathway fiber bundles were clearly revealed in 10 volunteers and 17 patients, and optic nerve was not wholly revealed in the rest of the subjects. Shift of optic nerve caused by compression and partial deformation were seen in 7 patients with orbital tumor. In 6 of 7 patients, DTI displayed significant abscise and deformation of visual nerve. Chi-square test indicated significant correlation between visual acuity decrescence and DTI visual nerve non-display.</p><p><b>CONCLUSIONS</b>Visual nerve fiber bundles and the whole visual pathway were visualized in most of patients with DTI. It might be an effective method of providing imaging evidence for visual nerve fiber earlier period functional changes, and laid a foundation for the study in other cranial nerves.</p>

The development and challenge of vision prosthesis / 中国医疗器械杂志

This paper introduces the current development and challenges of vision prosthesis.

Value of fast dynamic contrast enhanced MRI in the diagnosis of orbital lesions / 眼科

Objective To study the value of fast dynamic contrast enhanced MRI in the diagnosis of orbital occupying lesions. Design Retrospective ease series.Participants 21 patients with orbital occupying lesions.Methods All the 21 patients were performed on fast dynamic contrast enhanced MRI and were verified by pathology.The raw datum were processed by the software of GE Functool. Parameters such as time-intensity curve(TIC),time to peak(Tpeak),1 minute enhancement ratio(ERlmin)and maximum enhancement ratio(ERmax)were analyzed to study the characteristics of orbital diseases on fast dynamic contrast-enhanced MRI.Main Outcome Measures TIC,ERlmin,and ERmax.Results The characteristics of TIC between benign diseases and malignant tumors were different. Of the 16 cases of benign lesions,12 demonstrated as continuous increasing type,and of 5 cases of malignant lesions,3 cases of lym- phoma were all platform type.The ER1min of the malignant tumors(150.47?42.18)was higher than that of the benign lesions (101.37?43.02)(P=0.021).Cavernous hemangiomas had special progressing enhancing model.Conclusions Fast dynamic contrast enhanced MRI is valuable to distinguish malignant tumors from benign occupying lesions.(Ophthalmol CHN,2007,16:305-308)

Comparative proteomics of the kidney in lupus nephritis mouse / 基础医学与临床

Objective To study the differential proteome of kidney between lupus nephritis mouse and normal mouse.Methods The proteins of kidney were separated by two-dimensional gel electrophoresis(2-DE).The gels stained by silver were scanned by ImageScanner and analyzed by PDQuest software.Results About 573?52 and 658?43 protein spots were found in the three maps of control group and LN group respectively;the match ratio was 83% and 87% respectively.One hundred and fourteen spots were found increased that showed a two fold increase as comparing to control group.Conclusion A significant difference in protein expression of LN mouse kidney was found and may be related to the pathogenesis of LN.

Expression of the recombinant SARS coronavirus nucleocapsid protein in Pichia pastoris and identification of its bioactivity / 生物工程学报

The full length cDNA of SARS coronavirus nucleocapsid (N) protein was amplified by PCR and cloned into yeast expression vector pPIC3.5K to generate expression vector pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into P. pastoris (His- Mut+) by electroporation method. His+ Mut+ recombinant strains were screened on G418-RDB and MM/MD plates, and further confirmed by PCR. The influence of various inducing media, dissolved oxygen(DO) and the different final concentration of methanol was subsequently investigated. The results showed that the FBS medium was optimal for recombinant N protein expression and growth of the recombinant strain. The optimal final concentration of methanol is 1% (V/V), and the DO has a significant effect on recombinant N protein expression and growth of recombinant strain. The recombinant N protein expressed was about 6% of the total cell proteins, 410 mg/L of recombinant N protein and 45 OD600 were achieved in shake flask. Western-blot showed that the recombinant N protein had high specificity against mouse-anti-N protein-mAb and SARS positive sera, but had no cross-reaction with normal human sera. The result of scale-up culture in fermemtator demonstrated that 2.5g/L of recombinant N protein and the maximum cell 345 OD600 of were achieved, which was 6.1 times and 7.7 times higher than that in shake flask. So this study provide a basis for further researches on the early diagnosis of SARS and the virus reproduction and pathology reaction of SARS coronavirus.

Identification of a novel splice mutation of low density lipoprotein receptor gene in a Chinese family with familial hypercholesterolemia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify the mutation of low density lipoprotein receptor(LDLR) gene in a large Chinese family with familial hypercholesterolemia(F H) and make a discussion on the pathogenesis of FH at the molecular level.</p><p><b>METHODS</b>Investigations were made on a patient with the clinical phenotype of homozygous FH and his parents for mutations of promoter and all 18 exons of LDLR gene. Screening was carried out using Touch down PCR and a g arose gel electrophoresis, combined with DNA sequence analysis. The results were compared with the normal sequences in GenBank and FH database (www.ucl.uk/fh) t o find the mutation. Then the mutation was identified in other members of the family. In addition, the authors screened the apolipoprotein B(100) (apoB(100)) gene f or known mutations (R3500Q) that cause familial defective apoB(100) (FDB) by PCR-RFLP.</p><p><b>RESULTS</b>A novel homozygous IN III 5' GT --> AT mutation in the splice donor of LDLR intron 3 was detected in the homozygote propositus with FH. The mutation was also identified in four heterozygous carriers in his family. No mutations R3500Q of apoB(100)were observed.</p><p><b>CONCLUSION</b>A homozygous G --> A splice mutation in LDLR gene was first reported. The change of the splice donor in LDLR intron 3 may cause skipping of exon 3, which is responsible for FH. Perhaps it is a particular pathogenesis for Chinese people.</p>

Hepatitis E virus capsid protein production by high cell density culture of recombinant Escherichia coli / 生物工程学报

Production of Hepatitis E Virus capsid protein by high cell density culture in recombinant E. coli has been studied in 10L and 30L fermentors. The effects of different factors on growth and producing recombinant protein of E. coli have been studied by batch culture, such as different media, the ratio of phosphate and Magnesium sulfate. Comparison of fermentation performance for recombinant E. coli in different fed-methods culture has been investigated by fed-batch culture. The effects of inducing at different stages of growth and time of inducing on growth and producing recombinant protein, also obtained by fed-batch culture. At last, the solubility of inclusion body in different urea concentrations also has been obtained by fed-batch culture. The results show that the concentration of phosphate and Magnesium sulfate in the optimal media is 80mmol/L and 20mmol/L in batch culture respectively, that induction with 1.0mmol/L IPTG at mid log phase (about 45 OD at 600nm) is suitable for growth and recombinant protein expression, the cells were approaching stationary growth phase and the maximum cell OD at 600nm of 80 was achieved in 5h of fed-batch culture, and the expression level is 29.74%. The results also indicate that the solubility of inclusion body in 4mol/L urea solution induced at 37 degrees C reaches 14mg/mL, over 80% inclusion body was resolved. The culture process achieved in 10L fermentor could be successfully scaled up to 30L fenmentor with good reproducibility.

Interface domain of hepatitis E virus capsid protein homodimer / 生物工程学报

Hepatitis E is a main cause of acute viral hepatitis in developing countries where it occurs as sporadic cases and in epidemics form. The causative agent, hepatitis E virus, is transmitted primarily by the fecal-oral route. The approximately 7.5 kb positive-sense single-strand RNA genome includes three open reading frames (ORFs), one of which (ORF2) is postulated to encode the major viral capsid protein (pORF2) of 660 amino acid residues. We earlier showed that a bacterially expressed peptide, designated as NE2, located from amino acid residues 394 to 606 of ORF2, was found to aggregate into homodimer to at least hexamer. To understand the interface domains within this peptide vital for dimerization and formation of major neutralizing epitopes, NE2 protein underwent terminal-truncated and site-directed mutation. The hydrophobic region, ORF2 aa597-aa602 (AVAVLA), played a key role in oligomerization. Any amino acid residue of this region replaced with glutamic acid residue, the peptide can not refold as homodimer and/or oligomer. The immunoreactivities of these mutant peptides, blotted with anti-HEV neutralizing monoclonal antibody (8C11) and convalescent human sera, show associated to the formation of homodimer. The intermolecular contact region on homodimer was investigated by chemical cross-linking of two site-directed cysteines. When the alanine on aa597 site mutated with cysteine, two different homodimers were found in SDS-PAGE analysis. One (42kD) can be disassociated with 8mol/L urea, which is postulated to form by virtue of hydrophobic interaction, and the other (60kD) falls apart with the reductant DTT present. The exact conformation, generating the cross-linking reaction of cysteines, was further investigated by induced-oxidation on monomer and hydrophobic homodimer of A597C protein with GSH/GSSG. And the results revealed, it is the conformation of hydrophobic homodimer that induces the disulfide bond come into being, instead of the one of monomer. So the aa597 site was verified to be located on interface domain of hydrophobically interacting homodimeric complex. To evaluate the biological significance of hydrophobicity of interface domain, we searched natural variations as to the region on all available databases with NCBI blast program. All variations on these amino acid residues kept higher hydrophobicity, which suggests that the hydrophobic domain is critical for the assemblage and propagation of HEV. NE2 N-terminal deletions up to aa458 had no effect on dimerization and took no exact part in formation of major neutralizing epitopes, but the fragment may act as helper for the formation of major neutralizing epitopes on NE2. Interestingly, the C-terminus aa605-aa660 of ORF2 can also act as helper instead of the N-terminus of NE2. This study suggests an interface domain of NE2 might be vital for HEV capsomer assembly and formation of major neutralizing epitopes. These results may offer clues to the rational design of recombinant anti-HEV vaccine.

Particulate recombinant hepatitis E virus capsid protein and its antigenicity and immunogenicity / 生物工程学报

An E. coli expressed recombinant antigen NE2 was reported to aggregate into homo-oligomer, and can induce protective antibodies on rhesus monkey, but its immunogenicty was much weak after being purified. In this study, three N-terminal extension mutant of NE2 were expressed in E. coli, one of which named HEV 239 was found to aggregate into particle. HEV 239 antigen had good reactivity with sera of hepatitis E patients. The reactivity of HEV 239 against neutralization monoclonal antibody 8C11 was similar as NE2 antigen, while the reactivity of it against another neutralization monoclonal antibody 8H3 is much better than NE2 antigen, which indicated better antigenicity of HEV 239 than NE2. The diameter of purified HEV 239 particulate antigen was between 15 nm to 30 nm. The ED50 of immunization of HEV 239 particle adsorbed by aluminum adjuvant to BALB/c mice was between 0.08 microg to 0.25 microg. In contrast, the seraconversion rate of mice immunized by NE2 antigen adsorbed by aluminium adjuvant was only 25% on 60 microg vaccination. These results suggested that HEV 239 antigen particle has better immunogenicity as well as antigenicity than those of NE2 antigen, so it is a better vaccine candidate against HEV.

The study of aggregate of the ORF2 peptide of hepatitis E virus expressed in Escherichia coli / 生物工程学报

A fragment of hepatitis E virus open reading frame-2(ORF2), located from amino acid residues 394 to 604, was expressed in E. coli. The recombinant protein NE2 was found to form homodimer mostly in SDS-PAGE, which can be dissociated to monomers when treated with urea, and it was recognized more strongly in its dimeric form than the monomer by HEV reactive human serum in Western blotting. Besides, many aggregated form of NE2 from dimer to at least hexamer can be seen in MALDI-TOF-MS. And when the hydrated dynamic semidiameter of NE2 moleculars in PBS was measured as about 4 nm by Dynamic Light Scattering (DLS), being equal to tetramer, but with high polydispersity, which suggested that the NE2 moleculars were existed in PBS in many different sizes. These results suggested that the recombinant NE2 can aggregate into several oligomer forms, the association in the dimer is most strong, and dimers can assemble further to form some super-structure.