Analysis on characteristics and influencing factors of COVID-19 confirmed cases with viral nucleic acid re-positive after discharge in Guangdong Province / 中华预防医学杂志

Objective: To analyze the epidemiological characteristics and influencing factors of COVID-19 confirmed cases with viral nucleic acid re-positive in anal and/or throat swabs after discharge during the domestic imported epidemic stage in Guangdong Province in early 2020. Methods: The COVID-19 confirmed cases with the onset time before March 1, 2020 in Guangdong Province were collected to analyze the demographic data, epidemiological characteristics, and specimen collection and testing data after discharge. Logistic regression model was used for influencing factors analysis of re-positive cases. Results: A total of 1 286 COVID-19 confirmed cases were included, the M(Q1,Q3) of age was 44(32,58)years, 617 cases were male, 224 cases were re-positive in anal and/or throat swabs with the re-positive rate 17.42%. The M(Q1,Q3) of age of re-positive cases was 35(23, 50) years, which was younger than that of re-negative cases age was those 46(33, 59) years (P<0.001). With the increase of age, re-positive rate decreased (χ2trend=52.73, P<0.001). 85.27% (191/224) of re-positive cases were found in 14 d after discharge, the duration time of re-positive status was 13(7, 24) d, and 81.69% (183/224) of re-positive cases were re-tested negative in 28 d after re-positive date. No fever and other symptoms had been observed among re-positive cases during the whole follow-up. No secondary infectious cases had been found among close contacts after 14 d of centralized isolation and sampling screening. Univariate logistic regression model analysis revealed that the influencing factors of the re-positive cases included age, occupation, clusters, clinical types, and admission time. Multivariate logistic regression model analysis revealed that age was an independent risk factor. Conclusions: SARS-CoV-2 viral nucleic acid re-positive is found in COVID-19 confirmed cases after discharge in Guangdong Province. Most re-positive cases are confirmed among 14 d after discharge and re-test to negative among 28 d after re-positive date. Age is an risk factor for re-positive cases after discharge.

Follow-up study on height growth of preschool children with different stable physique / 中国学校卫生

Objective@#To understand the characteristic of height growth among preschool children with normal physique, overweight and obese, and in order to provide basis for proper physical growth intervention of preschool children.@*Methods@#Cluster sampling method was used and preschool children of kindergartens in 7 cities were selected, height and weight was measured, the information of birth date and sex were collected by parents’ questionnaires. The "WHO Child Growth Standards" was used for evaluate children’s height and body mass index. ANOVA analysis and Wilcoxon rank sum test were used for statistic analysis.@*Results@#There were 2 479 children who were normal weight, overweight and obese at baseline and were followed up for 2 years. The detection rate of <P25 height decreased by years(12.08%, 3.70%, 2.21%), and ≥P75 height evaluation increased among children with different physique(35.20%, 55.56%, 73.48%). Compared with normal weight children, overweight and obese children had higher average height, annual height growth value, detection rate of annual height growth value greater than 7 cm, detection rate of ≥P75 height and height annual increase rate.@*Conclusion@#Parents and practitioners in MCH should pay attention to the children’s height growth, especially on overweight and obese children. More in-depth research are needed to explore the relationship between children’s height and physique.

Near and long-term effects of acceptance and commitment therapy on patients with type 2 diabetes / 中国实用护理杂志

Objective:To observe the near-and long-term effects of acceptance and commitment therapy (ACT) on psychological flexibility, self-efficacy, and glycemic control in patients with type 2 diabetes.Methods:A total of 96 patients with type 2 diabetes admitted to Zhuzhou Central Hospital during the period from January to December 2017 were selected as subjects. According to the random number table method, they were divided into the control group and the ACT group, 48 cases in each group. The control group was given regular health education, and the ACT group was given ACT-oriented health education. The psychological flexibility, self-efficacy and glycemic control of the two groups were compared before intervention, 7 weeks after intervention and 1 year after intervention.Results:After 7 weeks of intervention, the psychological flexibility and self-efficacy scores of the ACT group was (21.47±4.89) and (8.96±1.70) respectively, the control group was (25.28±6.33) and (7.80±1.42) respectively. After 1 year of intervention, the psychological flexibility and self-efficacy scores of the ACT group was (23.87±5.03) and (8.09±1.38) respectively, and the control group was (27.19±5.48) and (6.97±1.24) respectively. The ACT group was significantly better than the control group, and the difference was statistically significant ( t=-3.300-4.044, P<0.01). After 7 weeks of intervention, the effective rate of blood glucose control in the ACT group was 93.75% (45/48), and the control group was 75.00% (36/48). After 1 year of intervention, the effective rate of blood glucose control in the ACT group was 86.96% (40/46), and the control group was 65.91% (29/44). The ACT group was significantly higher than the control group, and the difference was statistically significant ( χ2=6.400, 5.569, P<0.05). Conclusions:The application of health education activities based on ACT is significant in patients with type 2 diabetes, can significantly improve the patient's near-term psychological flexibility and self-efficacy, conducive to better long-term blood sugar management.

Regulation of γδT17 cells by Mycobacterium vaccae through interference with Notch/Jagged1 signaling pathway

The objective of this study was to investigate the effect of Mycobacterium vaccae on Jagged 1 and gamma delta T17 (γδT17) cells in asthmatic mice. An asthma mouse model was established through immunization with ovalbumin (OVA). Gamma-secretase inhibitor (DAPT) was used to block the Notch signaling pathway. M. vaccae was used to treat asthma, and related indicators were measured. Blocking Notch signaling inhibited the production of γδT17 cells and secretion of cytokine interleukin (IL)-17, which was accompanied by a decrease in Jagged1 mRNA and protein expression in the treated asthma group compared with the untreated asthma group. Similarly, treatment with M. vaccae inhibited Jagged1 expression and γδT17 cell production, which was associated with decreased airway inflammation and reactivity. The Notch signaling pathway may play a role in the pathogenesis of asthma through the induction of Jagged1 receptor. On the other hand, the inhibitory effect of M. vaccae on Jagged1 receptor in γδT17 cells could be used for the prevention and treatment of asthma.

Near and long-term effects of acceptance and commitment therapy on patients with type 2 diabetes / 中国实用护理杂志

Objective@#To observe the near-and long-term effects of acceptance and commitment therapy (ACT) on psychological flexibility, self-efficacy, and glycemic control in patients with type 2 diabetes.@*Methods@#A total of 96 patients with type 2 diabetes admitted to Zhuzhou Central Hospital during the period from January to December 2017 were selected as subjects. According to the random number table method, they were divided into the control group and the ACT group, 48 cases in each group. The control group was given regular health education, and the ACT group was given ACT-oriented health education. The psychological flexibility, self-efficacy and glycemic control of the two groups were compared before intervention, 7 weeks after intervention and 1 year after intervention.@*Results@#After 7 weeks of intervention, the psychological flexibility and self-efficacy scores of the ACT group was (21.47±4.89) and (8.96±1.70) respectively, the control group was (25.28±6.33) and (7.80±1.42) respectively. After 1 year of intervention, the psychological flexibility and self-efficacy scores of the ACT group was (23.87±5.03) and (8.09±1.38) respectively, and the control group was (27.19±5.48) and (6.97±1.24) respectively. The ACT group was significantly better than the control group, and the difference was statistically significant (t=-3.300-4.044, P<0.01). After 7 weeks of intervention, the effective rate of blood glucose control in the ACT group was 93.75% (45/48), and the control group was 75.00% (36/48). After 1 year of intervention, the effective rate of blood glucose control in the ACT group was 86.96% (40/46), and the control group was 65.91% (29/44). The ACT group was significantly higher than the control group, and the difference was statistically significant (χ2=6.400, 5.569, P<0.05).@*Conclusions@#The application of health education activities based on ACT is significant in patients with type 2 diabetes, can significantly improve the patient's near-term psychological flexibility and self-efficacy, conducive to better long-term blood sugar management.

Health-related quality of life and health-adjusted life expectancy among patients with chronic non-communicable diseases, in Guangdong province / 中华流行病学杂志

Objective@#To estimate the health-related quality of life (HRQOL) and health-adjusted life expectancy (HALE) which were associated with chronic non-communicable diseases (NCDs) in people from Guangdong province of China.@*Methods@#Data on both NCDs prevalence and EuroQol-5 Dimensions-3 Levels measured HRQOL were gathered from the Fifth National Health Survey in Guangdong province, 2013. Logistic regression model and multiple linear regression model were employed to explore the impact of NCDs on HRQOL. Life expectancy (LE) and HALE were used to evaluate the comprehensive impact of chronic diseases on population health.@*Results@#A total of 68 550 inhabitants were included in the analysis. Graded logistic regression showed that the impact of chronic diseases on all dimensions of quality of life was statistically significant after adjusting for social demographic characteristics. The greatest health impact was on the pain/discomfort health dimension [OR=4.48 (95%CI:4.20-4.77)], followed by anxiety/depression[OR=3.95 (95%CI: 3.62- 4.31)], daily activities [OR=3.69 (95%CI: 3.37-4.04)], mobility [OR=3.63 (95%CI: 3.34-3.94)]and ability on self-care [OR=3.30 (95%CI: 2.98-3.66)]. Losses of LE and HALE caused by NCDs were 12.7 and 14.6 years respectively while the overall expected gain was 3.8 years in HALE, when NCDs were taken away.@*Conclusions@#Our data showed that NCDs had shortened the healthy life span of patients through reducing the HRQOL and also causing heavy disease burden on both patients with NCDs and the communities. Health-care related policies on NCDs need to be developed, for the elderly, in particular.

Intranasal Immunization Using CTA1-DD as a Mucosal Adjuvant for an Inactivated Influenza Vaccine / 生物医学与环境科学（英文）

OBJECTIVE@#To evaluate the effect of intranasal immunization with CTA1-DD as mucosal adjuvant combined with H3N2 split vaccine.@*METHODS@#Mice were immunized intranasally with PBS (negative control), or H3N2 split vaccine (3 μg/mouse) alone, or CTA1-DD (5 μg/mouse) alone, or H3N2 split vaccine (3 μg/mouse) plus CTA1-DD (5 μg/mouse). Positive control mice were immunized intramuscularly with H3N2 split vaccine (3 μg/mouse) and alum adjuvant. All the mice were immunized twice, two weeks apart. Then sera and mucosal lavages were collected. The specific HI titers, IgM, IgG, IgA, and IgG subtypes were examined by ELISA. IFN-γ and IL-4 were test by ELISpot. In addition, two weeks after the last immunization, surivival after H3N2 virus lethal challenge was measured.@*RESULTS@#H3N2 split vaccine formulated with CTA1-DD could elicit higher IgM, IgG and hemagglutination inhibition titers in sera. Furthermore, using CTA1-DD as adjuvant significantly improved mucosal secretory IgA titers in bronchoalveolar lavages and vaginal lavages. Meanwhile this mucosal adjuvant could enhance Th-1-type responses and induce protective hemagglutination inhibition titers. Notably, the addition of CTA1-DD to split vaccine provided 100% protection against lethal infection by the H3N2 virus.@*CONCLUSION@#CTA1-DD could promote mucosal, humoral and cell-mediated immune responses, which supports the further development of CTA1-DD as a mucosal adjuvant for mucosal vaccines.

Efficient Humoral and Cellular Immune Responses Induced by a Chimeric Virus-like Particle Displaying the Epitope of EV71 without Adjuvant / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To eliminate the side effects of aluminum adjuvant and His-tag, we constructed chimeric VLPs displaying the epitope of EV71 (SP70) without His-tagged. Then evaluating whether the VLPs could efficiently evoke not only humoral but also cellular immune responses against EV71 without adjuvant.</p><p><b>METHODS</b>The fusion protein was constructed by inserting SP70 into the MIR of truncated HBcAg sequence, expressed in E. Coli, and purified through ion exchange chromatography and density gradient centrifugation. Mice were immunized with the VLPs and sera were collected afterwards. The specific antibody titers, IgG subtypes and neutralizing efficacy were detected by ELISA, neutralization assay, and EV71 lethal challenge. IFN-γ and IL-4 secreted by splenocytes were tested by ELISPOT assay.</p><p><b>RESULTS</b>HBc-SP70 proteins can self-assemble into empty VLPs. After immunization with HBc-SP70 VLPs, the detectable anti-EV71 antibodies were effective in neutralizing EV71 and protected newborn mice from EV71 lethal challenge. There was no significant difference for the immune efficacy whether the aluminum adjuvant was added or not. The specific IgG subtypes were mainly IgG1 and IgG2b and splenocytes from the mice immunized produced high levels of IFN-γ and IL-4.</p><p><b>CONCLUSION</b>The fusion proteins without His-tagged was expressed and purified as soluble chimeric HBc-SP70 VLPs without renaturation. In the absence of adjuvant, they were efficient to elicit high levels of Th1/Th2 mixed immune response as well as assisted by aluminum adjuvant. Furthermore, the chimeric VLPs have potential to prevent HBV and EV71 infection simultaneously.</p>

Expression and purification of the chimeric virus-like particles displaying enterovirus 71 epitopes / 中华实验和临床病毒学杂志

Objective@#To study the expression and purification of the chimeric virus-like particles displaying epitopes of EV71 as a candidate of enterovirus 71 gene recombined vaccinet.@*Methods@#The fusion protein hepatitis B core (HBc)-SP70 was constructed by inserting SP70 into the main immunogenic region of truncated hepatitis B core antigen (HBcAg) sequence, expressed in E. Coli, and purified through sonication, ion exchange chromatography, CsCl cushion centrifugation and density gradient centrifugation. Then its antigenicity was detected by ELISA and Western blot assay.@*Results@#Recombinant plasmid pHBc-SP70 was successfully constructed. And the soluble fusion protein was efficiently expressed induced by IPTG. The purity of the chimeric virus-like particles (VLPs) was up to 90% after the purification process described in method . The purified fusion protein HBc-SP70 could be spontaneously folded and assembled into empty virus-like particles and react with the monoclonal antibodies against HBc and SP70.@*Conclusions@#The chimeric VLPs displaying epitopes of EV71 were efficiently expressed and purified in E. Coli. with excellent antigenicity, which laid a foundation for evaluation of the immune effect evoked by this EV71 gene recombined vaccine.

Preparation of peptide mimotope-based diagnostic antigen of Epstein-Barr virus infection / 中华实验和临床病毒学杂志

Objective@#To prepare peptide minotope-based recombinant diagnostic antigen of Epstein-Barr virus (EBV) infection and evaluate its antigenicity preliminarily.@*Methods@#With Trx at the N-terminal and His tag at the C-terminal, the peptide minotope of EBV (GP125, F1, A2, A3C2) was expressed in Escherichia coli and purified by affinity and anion exchange chromatography (designated 'H58’); based on antigenicity of H58 identified by Western blotting (WB), we constructed and evaluated a novel early diagnostic ELISA for EBV infection.@*Results@#The soluble H58 protein with high concentration (2.8 mg/ml) and purity (99.01) was obtained; WB analysis found that there was an obvious band (28 ×103) on the NC membrane, using H58 anti-Trx monoclonal antibody or acute-phase sera of EBV infection as the first antibody. With the novel ELISA, 50 positive sera of EBV infection and 50 negative sera were detected, displaying that the grouping of OD value of positive serum (95%CI: 1.233-1.489) and negative serum (95%CI: 0.113-0.159) was different (P<0.05) with the sensitivity 98.0%, specificity 96.0% and kappa value 0.940.@*Conclusions@#By E. coli expression and affinity and ion exchange chromatography purification, the peptide minotope-based recombinant diagnostic antigen of EBV infection was obtained with excellent antigenicity, which could be applied for serological detection of EBV infection.

Expression and Purification of Recombinant Hepatitis Delta Virus (HDV) Antigen for Use in a Diagnostic ELISA for HDV Infection Using the High-Density Fermentation Strategy in Escherichia coli / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Hepatitis Delt a Virus (HDV) antigen is widely used as a capture antigen in ELISAs for the identification of HDV infection; large amounts of recombinant HDV antigen with active antigenicity are required for this purpose.</p><p><b>METHODS</b>Reconstruct the gene of HDV antigen based on the bias code of Escherichia coli, the recombinant protein expresses by high-density fermentation with fed-batch feeding strategy, and purify by immobilized metal chromatography. The sensitivity and specificity of this antigen detect by ELISA method.</p><p><b>RESULTS</b>The expression of HDV antigen can reach 20% of the total cell mass in the soluble form. The recombinant HDV antigen can be conveniently purified (98%) by immobilized metal ion affinity chromatography (IMAC) using the interaction between a His-tag and nickel ions. Production of recombinant HDV antigen can reach 0.5 g/L under conditions of high-density cell fermentation. Applied to the diagnostic ELISA method, the recombinant HDV antigen shows excellent sensitivity (97% for IgM and 100% for IgG) and specificity (100% for IgG and IgM) for the detection of anti-HDV antibodies.</p><p><b>CONCLUSION</b>Expression and purification the recombinant HDV antigen as a candidate protein for application in a diagnostic ELISA for HDV infection. Large-scale production of the protein can be achieved using the high-density fermentation strategy.</p>

Hepatitis B Immunoprophylactic Failure and Characteristics of the Hepatitis B Virus Gene in Mother-Infant Pairs in Parts of China / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To determine the hepatitis B immunoprophylactic failure rate in infants born to hepatitis B virus (HBV) infected mothers and to characterize HBV genes.</p><p><b>METHODS</b>HBV-serological testing was conducted for pregnant women and infants. The complete genomes of 30 HBV isolates were sequenced, and genetic characteristics were analyzed using MEGA 5 software.</p><p><b>RESULTS</b>The immunoprophylactic failure rate for infants who had completed the scheduled hepatitis B vaccination program was 5.76% (32/556). High sequence homology (99.8%-100%) was observed in 8 of the 10 mother-infant pairs. We identified 19 subgenotype C2 strains, 9 subgenotype B2 strains, and 2 subgenotype C1 strains. Three serotypes were detected: adr (19/30), adw (9/30), and ayw (2/30). The frequency of amino acid mutation of the 'a' determinant region was 16.67% (5/30), including that of Q129H, F134Y, S136Y, and G145E. We detected 67 amino acid mutations in the basal core promoter, precore, and core regions of the genome.</p><p><b>CONCLUSION</b>The immunoprophylactic failure rate in infants born to HBV-infected mothers is low in the regions of China examined during this study. Moreover, HBV mutation in the 'a' determinant region could not account for immunoprophylactic failure for all infants.</p>

A preliminary comparative study on two different methods of region of interest selection on the pyramidal tract tracking in the surgery of central area lesions / 中国神经精神疾病杂志

Objective To validate and compare the pyramidal tracts traced by functional magnetic resonance mo?tion activated area as the region of interest method (fMRI guided DTI-FT) and by the anatomy of primary motor cortex as region of interest method(traditional DTI-FT)using subcortical electrical stimulation (DsCS). Methods A prospective study was conducted in 12 cases of patients with central lesions involving the motor area. The pyramidal tracts were traced by fMRI guided DTI-FT method and traditional DTI-FT method. The lesions were resected with the assistance of neuronavigation. The distances between same stimulation positive point and pyramidal tracts traced by the fMRI DTI-FT or traditional DTI-FT were recorded. The coincidence rates between pyramidal tract imaging and DsCS were analyzed in order to verify the accuracy and reliability of these two methods. Results Two cases were excluded:one due to the failure of the fMRI activation caused by movement dysfunction and one case due to negative electrical stimulation.,The pyrami?dal tracts were successfully reconstructed in the rest 10 patients using these two methods which were further applied to assist surgery. The coincidence rates between DsCS and pyramidal tracts were 77%in fMRI DTI-FT and 70%in tradition?al DTI-FT. The shortest distances were 4.3mm±2.8mm and 5.5mm±3.4mm in fMRI DTI-FT and in traditional DTI-FT in 16 DsCS positive sites and the difference was statistically significant (P<0.05). Five cases had temporary postoperative pa?ralysis. Among them, four cases had upper limb paralysis and one case had hemiplegia. The motor function was improved in four cases and remained unchanged in two cases two weeks after the operation. The motor function in the rest six cases did not have any change before and after operation. Conclusion The fMRI guided DTI-FT can be helpful to deal with le?sions and effectively protect the brain function area in patients with the central area lesions involved motor area.

Cloning and expression and preliminary antigenicity identification for the diagnostic antigen of hepatitis E virus / 中华流行病学杂志

Objective To clone,express and purify thioredoxin (named as N5),a specific diagnostic antigen of hepatitis E virus (HEV),and to initially evaluate its antigenicity and serological test performance.Methods Based on the gene sequences of HEV-ORF2 and carboxyl terminal ORF3 on GenBank,the codon was optimized by the Escherichia coli codon preference,inserted it into prokaryotic expression vector M48 following total gene synthesization,and expressed in Escherichia coli fusion protein N5 recombined with Thioredoxin (TRX).Fusion protein was purified in affinity chromatography,evaluating its antigenicity with Western blot technology,then evaluating its serological test performance using the negative and positive serum samples confirmed of HEV infection with laboratory and clinical tests.Results The recombinant plasmid expressing N5 diagnostic antigen was successfully established; high-level expression and purification to obtain soluble diagnostic antigens; Western blot results indicating fusion protein N5 can be bound specifically with the serum of HEV IgM antibody positive,showing satisfactory antigencity; using fusion protein N5 as the capture antigen to build indirect ELISA,testing 40 serum samples of HEV cases confirmed by pathogen detection and clinical diagnosis and 40 serum samples of healthy people,with the sensitivity and specificity of 95％ (38/40) and 90％ (36/40) respectively.Conclusion Recombinant plasmid expressing the HEV diagnostic antigen recombined with thioredoxin was successfully established,and soluble fusion protein N5 was obtained with high expression and strong antigenicity,promising in its future applications.

Prokaryotic expression and characterization of two recombinant receptor-binding domain(RBD) proteins of human coronavirus NL63(HcoV-NL63) / 病毒学报

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.

Antigenic analysis of two chimeric hepatitis B core particles presenting the preS1 neutralizing epitopes / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct full-length hepatitis B core particles presenting preS1 aa 21-47 epitope and truncated core particles presenting preS1 aa 37-45 epitope on their surface and compare their antigenicity.</p><p><b>METHODS</b>PreS1 aa21-47 epitope and aa 37-45 epitope were inserted respectively into full-length hepatitis B core (aa 1-183) and truncated HBcAg (aa 1-144), between the 78th (Asp) and 79th (Pro). The genes synthesized after the codon optimization were ligated to the pET43. 1a vector with the same cohesive terminal (NdeI and XhoI) and expressed in the E. coli expression system. The morphology of the proteins of interest were observed by electron microscope and characterized by ELISA and Western Blotting.</p><p><b>RESULTS</b>The morphology of the virus-like particles were confirmed by electron microscope. H2 were solid particles with a diameter of (31.61 +/- 1.27) nm, while H3 were hollow particles with a diameter of (28.46 +/- 1.16) nm. Statistical analysis showed that H2 is larger than H3 in the diameter (P < 0.01). The antigenicity of the inserted epitopes and carrier protein were identified by ELISA and Western Blotting.</p><p><b>CONCLUSION</b>Chimeric hepatitis B core particles presenting the preS1 neutralizing epitopes on their surface have been expressed, purified and identified, which lays the foundation for its application in vaccine research.</p>

Construction of recombinant CHO cell strain for high expression of HBsAg / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To overexpress hepatitis B virus S gene in CHO cells cultured in serum-free media.</p><p><b>METHOD</b>Plasmid was constructed by cloning of HBV S gene and then it was transfected into CHO cells. After cell screen, the positive clones were identified and isolated into a serum-free media followed by the serological and morphological characterization of the expression product.</p><p><b>RESULT</b>CHO cell strains which can express HBsAg efficiently and stably were obtained. Spherical and filamentous HBsAg could be detected under electronic microscope. The titer of the expression product was up to 1:5000.</p><p><b>CONCLUSION</b>Serum-free media cultured CHO cell strain for overexpression of HBsAg was successfully constructed and the expression product was high antigenic.</p>

Prokaryotic soluble expression of protein D of Haemophilus influenzae type b / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To express the recombinant D protein in prokaryotic expression system solubly and make preparation for producing D-carrier conjugate vaccine next step.</p><p><b>METHODS</b>The hpd gene fragment removed of signal peptide from genomic DNA of Hib CMCC was inserted into pET43. 1a. The recombinant plasmid was transformed to competent E. coli BL21 (DE3) for expression under induction of IPTG. The expressed recombination protein was precipitated with ammonium sulfate, purified by DEAE anion exchange column chromatography and identified for reactogenicity by Western Blot.</p><p><b>RESULTS</b>The expressed recombination protein, in a soluble form, constained about 50% of total somatic protein and showed specific reaction with the HIB antisera after preliminary purification.</p><p><b>CONCLUSION</b>The D protein recombined expression plasmid was constructed successfully and expressed D protein in prokaryotic cells in a solube form.</p>

Gene optimization, protein expression, purification and characterization of the HDV antigen for diagnosis / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare HDAg with biological activities as a candidate of diagnostic reagent.</p><p><b>METHODS</b>To synthesize HDV gene fragment after codon optimization. To construct a thio-fused recombinant plasmid based on M48 expression vector. To express in E. coli induced by IPTG. To purify the protein by affinity chromatography followed by characterization in ELISA:</p><p><b>RESULTS</b>Plasmid construction was verified by enzyme digestion. SDS-PAGE indicated the molecular weight of the protein was the same as we expectation. ELISA proved its affinity with HDV antibodies.</p><p><b>CONCLUSION</b>HDAg was obtained successfully and it will pave the road to the research of HDV diagnostic reagent.</p>

The assemblage, purification and characterization of EV71 VLPs expressed in baculovirus / 病毒学报

To construct a recombinant expression plasmid Bacmid-P1-3CD containing the P1 and 3CD genes of enterovirus 71(EV71), the P1 and 3CD genes were cloned into the same baculovirus shuttle vector (Bacmid). Recombinant AcMNPV-P1-3CD was obtained by transfecting the Bacmid-P1-3CD into the insect cell line of S f9. With the IFA and Western-blot methods for identification of expression products confirmed that the target protein was expressed in interior of infected S f9 cells. Electron microscopy showed that the structural protein capsid P1 was cut by virus-encoded protease 3CD and assembled into EV71 virus like particles (VLPs) about 27nm diameter. Different values of MOI and time points of expression were compared to explore the optimal expression condition, and the results showed that the time point could be a more important factor. Then we used S f9 cells with serum-free medium in CellSTACK-10 Culture Chambers to produce EV71 VLPs in the confirmed condition. After purification of VLPs by density gradient centrifugation, we observed on SDS-PAGE profile the purified sample contained three major proteins whose molecular masses corresponded to those of VP1 (39kD), VP0 (34kD) and VP3 (26kD) as well as the intact structure, which can be greatly used for further study in protein structure and genetic engineering vaccine research.