Diversity and recombination of human parechovirus in children with acute gastroenteritis in Guangzhou, China.

Human parechovirus (HPeV), a member of Picornaviridae family, is a widespread pathogen causing a wide spectrum of diseases. Like other picornaviruses, HPeV genome recombination has been detected. A total of 322 fecal samples were collected from children outpatients in Guangzhou, China, including 42 (13.0%, 42/322) HPeV-positive samples detected in most of the infected children less than two years old. Seven HPeV genotypes (HPeV1, HPeV3, HPeV4, HPeV5, HPeV6, HPeV8 and HPeV14) were detected, among which, HPeV14, a rare genotype, was reported for the first time in children with acute gastroenteritis in China. This study revealed recombination events in eight samples. Clinical profiles did not yield statistical significance between children with HPeV infection alone and cases without pathogens detected. In conclusion, this study demonstrated that HPeV circulated in Guangzhou, China is diverse genetically, which provided evidence of recombination in HPeV in China.

High prevalence of thalassemia in migrant populations in Guangdong Province, China.

BACKGROUND: The objectives of this study were to estimate the prevalence of thalassemia and to analyze the need for public health services for migrant populations in different cities in Guangdong Province, China. METHODS: A cross-sectional survey was conducted in 21 cities of Guangdong Province. Twenty-three types of a- and ß-globin gene mutations were detected in a total of 14,230 pregnant women and 14,249 husbands. RESULTS: There was a 16.45% prevalence of thalassemia among the 28,479 individuals, and the prevalences of α-, ß-, and combined α-/ß- thalassemia were 12.03%, 3.80%, and 0.63%, respectively. Compared with the native city residents in the province, the migrants from within the province and the immigrants from outside the province had lower prevalences of thalassemia, but the prevalence values were >11%. CONCLUSIONS: The prevalence values for thalassemia gene mutations were high in all three population groups studied in Guangdong Province. The results indicate that all segments of the Guangdong population should be screened for thalassemia.

Effect of resveratrol on gliotransmitter levels and p38 activities in cultured astrocytes.

Accumulating evidence suggests that resveratrol may have beneficial effects against traumatic brain injury. However, its effect on the regulation of extracellular levels of gliotransmitter and on the activation of p38 MAPK in astrocytes is still unknown. We have examined whether resveratrol regulates extracellular levels of gliotransmitter as well as the activation of p38 MAPK in cultured astrocytes before and after stretch injury. The extracellular levels of glutamate, D-/L-serine and D-serine were apparently reduced by 100 µM resveratrol in control astrocyte cultures. The dramatic increase of glutamate and D-serine release induced by stretch injury was also clearly inhibited by resveratrol. Resveratrol mediates this response by reduction of release through inhibition of extracellular calcium influx and increment of gliotransmitter uptake through enhancement of amino acid transporter expressed in the membrane of astrocyte. In addition, resveratrol definitely reduced the activation of p38 MAPK in cultured astrocytes following stretch injury. AMPA receptor is involved in the activation of p38 following injury. Conversely, the levels of glutamine and glycine were not obviously affected by resveratrol before and after injury. Intracellular levels of glutamate and D-serine are not apparently changed by stretch injury. Collectively, our data suggest that resveratrol might play an important role in protection of the nervous system after injury by decreasing the extracellular levels of gliotransmitter and inhibiting activation of p38 MAPK following injury.

Surveillance and risk factors of norovirus gastroenteritis among children in a southern city of China in the fall-winter seasons of 2003-2006.

AIM: Noroviruses (NoVs) are an important cause of acute gastroenteritis but knowledge on the disease burden and epidemiology in children in the developing countries remains limited. In this study, we performed a surveillance of NoV gastroenteritis in children of China to address some of the questions. METHODS: Faecal specimens from children (<5 years of age) at outpatient clinics of the Nan Fang Hospital in Guangzhou, China during the fall-winter seasons in 2003-2006 were tested for rotaviruses (RVs) and NoVs. A questionnaire on clinical records and hygiene habits was collected from each patient. RESULTS: Among 957 stool specimens tested, 488 (51%) specimens were positive for RVs. NoVs were detected in 112 (24%) of the 469 RV negative specimens. The Genogroup II (GII), particularly GII-4, viruses were predominant. No significant difference of clinical symptoms, hospitalisation and patient care expenses were found between children infected with NoVs and RVs. Consumption of uncooked food is a risk for NoV infection. Contact with diarrhoea patients is a suspected risk factor. Cutting nails frequently is a protective factor against NoV infection. CONCLUSIONS: NoVs are an important cause of acute gastroenteritis in children which need special attention of patient care at the clinics in addition to RVs. The awareness of those risk factors may help future disease control and prevention.

Synovial Fluid Eotaxin-1 Levels May Reflect Disease Progression in Primary Knee Osteoarthritis Among Elderly Han Chinese: A Cross-Sectional Study.

OBJECTIVE: The CC chemokine family member eotaxin-1, also named chemokine C-C motif ligand 11 (CCL11), has been detected in knee osteoarthritis (OA) and could induce breakdown of cartilage matrix. This study was performed to investigate the plasma and synovial fluid eotaxin-1 levels with the disease progression in elderly Han Chinese with primary knee OA. DESIGN: A total of 143 elderly primary knee OA patients and 135 healthy controls were enrolled in the study. The Western Ontario and McMaster Universities Arthritis Index (WOMAC) was performed to evaluate the clinical severity. The radiographic severity was assessed by Kellgren-Lawrence (K-L) grading. Plasma and synovial fluid (SF) eotaxin-1 levels were explored using enzyme-linked immunosorbent assay. The SF levels of matrix metalloproteinase-3 (MMP-3) and interleukin-6 (IL-6) were also examined. RESULTS: Elevated plasma eotaxin-1 levels were found in knee OA patients compared with healthy controls. Eotaxin-1 levels in SF of knee OA patients with K-L grade 4 were significantly elevated compared with those with K-L grades 2 and 3. Meanwhile, knee OA patients with K-L grade 3 had significantly increased SF levels of eotaxin-1 compared with those with K-L grade 2. Plasma eotaxin-1 levels in different K-L grading did not reach significant difference. Eotaxin-1 levels in SF of knee OA patients were significantly associated with disease severity evaluated by KL grading criteria. In addition, eotaxin-1 levels in SF were positively related to clinical severity illustrated by WOMAC as well as biochemical markers MMP-3 and IL-6. CONCLUSIONS: Eotaxin-1 levels in SF instead of plasma, were independently and positively related to the disease severity in elderly knee OA patients. The inhibition of eotaxin-1 and its related signaling pathways may serve as a novel therapeutic approach for OA progression.

[Ureaplasma urealyticum infection in the genital tract reduces seminal quality in infertile men].

OBJECTIVE: To understand Ureaplasma urealyticum (Uu) infection, analyzed the influence of Uu infection on the seminal quality and the accessory genetical gland function in male infertility patients, and investigate its mechanism. METHODS: We cultured 202 semen samples collected from male infertility patients and analyzed the influence of Uu infection on seminal parameters and the biochemical indexes of the seminal plasma. RESULTS: The Uu infection rate was 33.7% in the infertile males, with no statistic differences between the Uu positive and negative groups either in the average age (28.9 +/- 4.7 yrs vs 29.6 +/- 4.0 yrs, P = 0.250) or in the seminal quantity (2.93 +/- 1.32 ml vs 2.86 +/- 1.52 ml, P = 0.774). The sperm density, motility and vitality were (84.37 +/- 52.92) x 10(6) ml, (44.62 +/-22.13) % and (38.40 +/- 15.61) % in the Uu positive group, significantly lower than (101.90 +/- 43.90) x 10(6) ml, (51.83 +/- 19.88) % and (44.45 +/- 15.47) % in the Uu negative group (P = 0.025, P = 0.036 and P = 0.020). The seminal pH value was normal in both of the groups, but significantly higher in the Uu positive than in the negative group (7.32 +/- 0.10 vs 7.19 +/- 0.29, P = 0.003). VCL, VSL, VAP and MAD were significantly lower, while BCF was significant higher in the former than in the latter [(33.97 +/- 8.96) microm/s vs (39.70 +/- 8.14) microm/s, t = 4.113, P < 0.001; (22.29 +/- 6.06) microm/s vs (25.20 +/- 6.67) microm/s, t = 2.684, P = 0.008; (25.96 +/- 6.83) microm/s vs (30.02 +/- 6.81) microm/s, t = 3.537, P < 0.001; 46.60 +/- 13.68 vs 54.23 +/- 15.14, t = 3.112, P = 0.002; (6.12 +/- 1.89) Hz vs (5.22 +/- 1.64) Hz, t = 3. 164, P = 0.002]. All the five indexes were influenced by Uu infection. Compared with the negative group, the seminal plasma alpha-glucosidase was significantly decreased in the positive group [(40.0 +/-18.7) U/ml vs (47.9 +/- 21.0) U/ml, t = 2.248, P = 0.026], and the risk of the decrease was 2.12 times higher. No statistic difference was observed in seminal plasma acid phosphatase and seminal plasma fructose between the two groups. CONCLUSION: Uu infection in the genital tract is an important factor of seminal quality reduction in infertile men and may cause a decreased secretion of alpha-glucosidase in the epididymis, but it hardly influences the prostate and seminal vesicle.

Specific detection of toxigenic vibrio cholerae based on in situ PCR in combination with flow cytometry.

OBJECTIVE: To develop an in situ PCR in combination with flow cytometry (ISPCR-FCM) for monitoring cholera toxin positive Vibrio cholerae. METHODS: In running this method, 4% paraformaldehyde was used to fix the Vibrio cholerae cells and 1 mg/mL lysozyme for 20 min to permeabilize the cells. Before the PCR thermal cycling, 2.5% glycerol was added into the PCR reaction mixture in order to protect the integrality of the cells. RESULTS: A length of 1037bp DNA sequence was amplified, which is specific for the cholera toxin gene (ctxAB gene). Cells subjected to ISPCR showed the presences of ctxAB gene both in epifluorescence microscopy and in flow cytometric analysis. The specificity and sensitivity of the method were investigated. The sensitivity was relatively low (10(5) cells/mL), while the specificity was high. CONCLUSION: We have successfully developed a new technique for detection of toxigenic Vibrio cholerae strains. Further study is needed to enhance its sensitivities. ISPCR-FCM shows a great promise in monitoring specific bacteria and their physiological states in environmental samples.

[Effect of Fructus Bruceae oil emulsion on cellular immune function and quality of life in patients with non-small cell lung cancer].

OBJECTIVE: To observe the effect of Fructus Bruceae oil emulsion (FBE) on cellular immune function (CIF) and quality of life (QOF) in patients with non-small cell lung cancer (NSCLC) after chemotherapy. METHODS: One hundred and fifteen patients with mid-late stage NSCLC were randomly assigned to two groups, the 57 patients in the control group were only treated with chemotherapy of GP regimen, 58 in the treatment group with the chemotherapy of the same regimen and combined with FBE. The clinical efficacy was evaluated after two cycles of chemotherapy. RESULTS: The effective rate was 51.8% and 47.4% in the treatment group and the control group respectively, the difference between them was insignificant (P > 0.05). CIF and QOF in the treatment group were better than those in the control group after chemotherapy respectively (P < 0.01), in the latter, CIF and QOF were desreased after chemotherapy (P < 0.05). CONCLUSION: FBE combined with chemotherapy can improve the cellular immune function and quality of life in patients with mid-late stage NSCLC.

Enteroaggregative Escherichia coli isolated from Chinese diarrhea patients with high-pathogenicity island of Yersinia is involved in synthesis of siderophore yersiniabactin.

AIM: To investigate the distribution of 12 high-pathogenicity island (HPI) genes and the relation between HPI genes and expression of yersiniabactin (Ybt) in enteroaggregative E.coli (EAggEC) isolated from Chinese diarrhea patients. METHODS: The distribution of 12 HPI genes was investigated by PCR and DNA hybridization in two prototype strains of EAggEC, EAggEC 17-2, EAggEC O42, and 6 clinical EAggEC isolates from China. The production of siderophore Ybt in HPI-positive strains was detected by reporter gene bioassay to determine the relation between HPI genes and expression of Ybt. Flow cytometry was used to detect fluorescent signal of the reporter strain that could designate production of Ybt. RESULTS: Seven strains were HPI-positive and one strain was HPI-negative. Six of seven HPI-positive strains were inserted into asnT-tRNA site. Moreover, seven EAggEC HPI-positive strains revealed enhanced fluorescence signal but the EAggEC HPI-negative strain did not. However, there was a difference in Ybt expression condition and level among these seven EAggEC HPI-positive strains. Although UFT073 strain, the prototype strain of uropathogenic E.coli (UPEC), carried the complete HPI core part, we did not detect the expression of Ybt in it. CONCLUSION: EAggEC HPI-positive strains can express the Ybt system, but the presence of HPI core part does not mean the functional expression of Ybt.

[A questionnaire-based survey of the quality of life of 783 air-force soldiers and officers in the southern military region of China].

OBJECTIVE: To investigate the quality of life of the air-force soldiers and officers in the southern military region of China. METHODS: A self-rated health measurement scale was used to evaluate the quality of life of 783 air-force soldiers and officers in view of their physical, mental and social statuses. RESULTS: The quality of life of the subjects was at an intermediate level. Their physical, mental and social scores were 66.33+/-9.38, 58.99+/-10.09 and 53.69+/-12.40, respectively, with a total score of 178.44+/-22.60. The quality of life of the soldiers and officers significantly differed with educational background (P<0.01), places of enlisting (P<0.01), arm of the service (P<0.05), identities or positions (P<0.01), and occupation before enlisting (P<0.01). CONCLUSIONS: The quality of life of the soldiers and officers can be affected by such factors as educational back- ground, places of enlisting, arm of the service, identities or positions, and occupation before enlisting, but the nationalities or status of being the only child in family does not obviously influence the quality of life of the military personnel.

[Summary of Guangdong provincial seminar on avian influenza and influenza].

On 8th November 2005, an academic seminar on avian influenza and influenza in Guangdong Province was held by Guangdong Society of Tropical Medicine and the Epidemiology Committee of the Guangdong Preventive Medicine Society in Southern Medical University, addressing the current problems in epidemics of avian influenza. The specialists attending the conference arrived at the common consideration that at present, the avian influenza virus H5N1 has not the capacity to trigger an pandemic in human population, but scattered cases had been reported to increase the suspicions of H5N1 virus transmission between humans. Due attention should be paid to the tendency of expansion of the host range and epidemic area, and the possibility of disastrous influenza pandemic among human populations persists, for which rational consideration is called for, and the role of specialists should be fully recognized who are endeavoring to examine the possible scale of influenza occurrence and devise strategy to deal with the epidemic in Guangdong province according to the practical situation in China. Increased funds and investment in scientific research on avian influenza is urged for influenza prediction and surveillance, rapid and early diagnostic assays, understanding of virus variation, mechanism of H5N1 virus adaptation to human hosts, effective medicines and vaccines for prevention and therapy of avian influenza. Laboratory bio-safety control should be enforced to prevent infections originated from laboratories. The specialists appeal that the media report the news objectively and issue the public warnings against avian influenza after consulting specialists, so as to avoid unnecessary social panic.

[Construction and characterization of mutant Enteroaggregative E. coli O42 strain with high-pathogenicity island deletion].

OBJECTIVE: To construct and characterize a mutant Enteroaggregative E. coli(EAggEC) O42 strain with in-frame deletion of high-pathogenicity island (HPI). METHODS: The kanamycin resistance gene (kan) was inserted between the sequences of irp8 and irp5 genes as the two homologous sequences for construction of the recombinant plasmid pCO85 by subcloning the recombined sequence into the suicide vector pCVD442. By homologous recombination and conjunction mobilization, EAO85 mutant with deletion of the core region of HPI about 24 kb spanning from irp8 to irp5 sequences was screened. RESULTS: Irp8 and irp5 genes of EAggEC O42 were exchanged by pCO85 by conjunction mobilization with the selection by sucrose. The EAO85 mutant was identified by their failure to yield PCR products with primers specific for the internal regions of HPI. CONCLUSION: We have successfully constructed a mutant of EAggEC O42 strain with HPI in-frame deletion, which may facilitate the exploration of the role of HPI in EAggEC strain.

Acute diarrhea during army field exercise in southern China.

AIM: During emergency period, infectious diseases can be a major threat to military forces. During field training in southern China, diarrhea is the main cause of nonbattle injury. To evaluate the causes of and risk factors for diarrhea in emergency period, we collected clinical and epidemiological data from the People's Liberation Army (PLA) during field training in southern China. METHODS: From September 25 to October 2 1997, 2636 military personnel were investigated. Fecal sample cultures for lapactic pathogens were obtained from 103 military personnel with diarrhea. In addition, a questionnaire was administered to 103 cases and 206 controls to evaluate the association between illness and potential risk factors. At the same time, another questionnaire of 1:4 case-case control was administered to 22 severe cases (each severe case paired 4 mild cases). RESULTS: The training troop's diarrhea incidence rate was significantly higher than that of garrison. The diarrhea incidence rate of officers was significantly lower than that of soldiers. A lapactic pathogen was identified in 63.1% (65/103) of the troops with diarrhea. Enterotoxigenic Escherichia coli (35.0%) and plesiomona shigelloides (16.5%) were the most common bacterial pathogens. All bacterial isolates were sensitive to norfloxacin and ceftazidine. However, almost all of them were resistant to sulfamethoxazole, trimethoprim-sulfamethoxazole, oxytetracycline, doxycycline, furazolidone, ampicillin and cloromycetin to a different degree. Risk factors associated with diarrhea included drinking raw water, eating outside, contacting diarrhea patients, lacking sanitation, depression, lacking sleep, which were established by multiple-factor logistic regression analysis. In addition, the unit incidence rate was associated with the density of flies and the average daily boiled water available by regression and discriminate analysis. CONCLUSION: A series of risk factors are associated with the incidence rate of diarrhea. Our results may provide a useful basis for prevention and cure of diarrhea in emergency period of PLA.

[In vitro selection and affinity function of the aptamers to Bacillus anthracis spores by SELEX].

To obtain oligonucleotide aptamers, specifically binding to Bacillus anthracis spores, and to find the relationship between the structures and the affinities, and to determine whether the aptamers can be used as a novel molecule for spore detection, a synthetic 35 mer random DNA library was subjected to 18 rounds of selection by using SELEX (systematic evolution of ligands by exponential enrichment) protocol against spores of Bacillus anthracis vaccine strain A. 16R. The selected aptamers were cloned and sequenced. Software packages CLUSTALX (1.8) and DNASIS v2.5 were employed to analyze the conserved sequences and second structures of the aptamers, respectively. Affinities of aptamers to the spores were visualized by biotin streptavidin horseradish peroxidase system. DAB was used to visualize signals, as an assay method. A membrane-based hybrid sandwich assay was developed for detecting Bacillus anthracic spores by using a 5'-biotinylated ssDNA aptamers and anti-spore antibodies. PCR amplification band pattern of the first round selection was different from that of the ninth round. The binding assay demonstrated that the affinity of the eighteenth round pool increased thirty-seven folds more than that of the first round pool. The affinities of the aptamers were different: the highest A at 450 nm was 1.20, and the lowest was 0.20. The secondary structure analysis revealed possible stem-loop and hairpin structures for binding to the spores. The colorimetry on the immuno-membrane got the best signal with a ratio of 16 microgram aptamer to 4x10(7) spores. A set of aptamers with considerable binding affinity to Bacillus anthracis spores was successfully selected from the initial random ssDNA pool. The stem-loop and hairpin at 5' end of the aptamers worked as the main motif in the interaction between oligonucleotides and spores, while the neighbor bases of the triple structure might affect the stability. Therefore ssDNA aptamers seem to be a type of potential diagnostic molecule.

[An epidemiologic investigation on infection with severe acute respiratory syndrome coronavirus in wild animals traders in Guangzhou].

OBJECTIVE: To investigate status of infection with severe acute respiratory syndrome coronovirus (SARS-CoV) in traders of wild animals wholesale markets in Guangzhou. METHODS: Serum antibody against SARS-CoV IgG was determined cross-sectionally and symptoms of respiratory infection were investigated retrospectively for part of traders of three wholesale markets for wild animals in Guangzhou. RESULTS: Overall rate of infection with SARS-CoV in 635 traders was 16.69%, varying in three different markets. Infection rate in market A mainly engaging in wild animals ranked the highest of 25.61%, significantly higher than that in markets B and C engaging in domestic fowls and snakes. Infection rate in traders only engaging in civet cats was 58.54%, significantly higher than that in traders engaging in snakes only (9.46%). In market A, infection rate varied in different persons, 59.34%, 20.59%, 16.00%, 15.22%, 10.40% and 9.68% in traders engaging in wild animals, managers, children of the traders, traders engaging in domestic fowls, traders engaging in snakes, and traders engaging in frozen food, respectively, in a decreasing pattern as their contact opportunities. During the period of SARS epidemic, detection rate of SARS-CoV antibody in people with symptoms of acute respiratory infection was higher (30.70%) than that in those without such symptoms (20.08%). Prevalence of symptoms of acute upper respiratory infection in people with positive antibody against SARS-CoV was higher (49.28%) than that in those with negative antibody (30.35%). CONCLUSIONS: Infection with SARS-CoV in traders of animal markets possibly related to their direct exposure to wild animals, particularly to civet cats. During the period of SARS epidemic, some of the traders did infect with SARS-CoV, but they were neglected due to clinically inapparent manifestations.

gyrA gene mutations in quinolone-resistant Shigellae flexneri.

OBJECTIVE: To study the resistance mechanism of quinolone against Shigellae flexneri. METHODS: The N-terminal coding region of gyrA gene of 38 quinolone-resistant Shigellae flexneri were amplified by PCR and mutations detected by single- strand conformation polymorphism (SSCP), followed by sequence analysis. RESULTS: Eight isolates were found to be positive for gyrA mutations, and DNA sequence analysis of gyrA gene revealed 2 mutations that resulted in changes of the amino acids (Ser-83--Leu, Asp-87--Gly). CONCLUSION: gryA gene mutations is closely related to high resistance of Shigellae flexneri to quinolone.

[Construction of plant expression vectors containing the gene encoding cholera toxin B subunit].

OBJECTIVE: To construct the plant expression vector containing the nucleotide sequence encoding cholera toxin B (CTB) subunits. METHOD: Using high-fidelity PCR, we amplified CTB genes that were then subcloned into the transition vector pRTL2. Following confirmation of the CTB nucleotide sequence, the vector was subcloned into the plant vector pBI121 that was subsequently transferred into Agrobacterium tumefaciens LBA4404 by electroporation. RESULTS: CTB DNA that was ligated into the transition vectors resulted in the 2 vectors designated as pRCTB and pRCTBK. After the 2 vectors were ligated into the plant binary vector pBI121 respectively, new plant binary vectors, namely pBI-CTB and pBI-CTBK, were produced. Analysis with restriction endonucleases confirmed successful transfer of pBI-CTB and pBI-CTBK into Agrobacterium tumefaciens LBA4404. CONCLUSION: With appropriate technological strategy, the plant binary expression vectors encoding CTB have been constructed, which facilitates further investigation of CTB protein expressions in transgenic plant.

Detection of the high-pathogenicity island of Yersinia enterocolitica in enterotoxigenic and enteropathogenic E.coli strains.

OBJECTIVE: To describe the distribution of high-pathogenicity island (HPI) of Yersinia enterocolitica in enterotoxingenic E.coli (ETEC) and enteropathogenic E.coli (EPEC), and to understand the structure and function of HPI. METHODS: PCR was used to detect irp2, fyua and asn-intB genes with subsequent sequence analysis of these genes. Nucleic acid in situ hybridization was employed to identify the specificity of irp2 and fyua. RESULTS: Thirty irp2-positive strains were isolated from 93 ETEC strains and 3 from 10 EPEC strains, making a positivity rate of 32.25% and 30% respectively, and the positivity rates of fyua gene in ETEC and EPEC were 21.51% and 30% respectively. In most of these positive isolates, HPI was bordered by an asn tRNA locus, as in Yersinia sp. CONCLUSIONS: This study demonstrates that the high positivity rate of HPI of Yersinia enterocolitica in ETEC and EPEC strains may be crucial to the virulence changes, virulence evolution and virulence regulation in E.coli.

Expression and purification of invasion plasmid antigen C of Shigella flexneri.

OBJECTIVE: To induce the expression of and purify invasion plasmid antigen C (IpaC) of Shigella flexneri for studying the pathogenesis of Shigella flexneri. METHODS: Prokaryotic expression plasmid pET32a-ipaC was constructed and incorporated into E.coli BL21 (lambda DE3). The engineered bacteria were induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) to express IpaC, which was identified by SDS-PAGE and purified by QIA expressionist system. RESULTS: SDS-PAGE presented a band for the fusion protein with the relative molecular mass of approximately 63 000, whose expression reached up to 11% of the total protein of E.coli BL21(lambda DE3). After proper purification, a purity of the target fusion protein of over 90% was achieved when the concentration of imidazole for elution was 350 mmol/L. CONCLUSION: The recombinant plasmid pET32a-ipaC has been stably and efficiently expressed in E.coli BL21 (lambda DE3), and QIA expressionist purification system proves to be simple and highly efficient.

Functional change of human peripheral blood monocyte-derived dendritic cells after recombinant adeno-associated virus type 2-mediated HBsAg gene infection.

OBJECTIVE: To observe the changes in the functions of human peripheral blood monocyte-derived dendritic cells (DCs) after hepatitis B virus surface antigen (HBsAg) gene infection mediated by recombinant adeno-associated virus type 2 (rAAV). METHODS: The levels of both interleukin (IL)-12 in the supernatant of in vitro cultured DCs infected with rAAV-HbsAg and interferon gamma (IFN-gamma) in the supernatant of the lymphocyte populations co-cultured with DCs were determined by ELISA. The functions of the rAAV-HbsAg-infected DCs were assessed by mixed leukocyte reaction (MLR), and the changes of the surface markers (including CD80, CD83, CD86 and HLA-DR) in response to the infection were detected by flow cytometry. RESULTS: After rAAV-HBsAg infection, the IL-12 secretion of DCs was significantly enhanced (P <0.05), while IFN-gamma production by the lymphocyte populations co-cultured with rAAV-HbsAg-infected DCs was reduced (P <0.01). rAAV-HBsAg infection of DCs did not affect the surface marker expressions and stimulation ability of the DCs in allogeneic lymphocytes reaction. CONCLUSION: DCs infected by rAAV-HBsAg are more efficient than naive DCs in eliciting the differentiation of the lymphocytes toward T helper type I cells, but the functions and the surface markers of DCs remain unaffected after the infection, suggesting the applicability of rAAV as a potentially useful vector for HBsAg gene transfer into the DCs for HBV immunotherapy.