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Objective Different library construction methods, combined with high-throughput sequencing methods, were used to understand the genetic characteristics of the virus of the first confirmed imported monkeypox case in mainland China, and to compare the advantages and disadvantages of different library types. Methods The herpetic fluids and nasopharyngeal swabs of the first imported monkeypox case in the Chinese mainland were used as samples. After nucleic acid extraction and quantification, the library was directly constructed or the amplicon library was constructed using the monkeypox virus whole genome capture kit, and the whole genome sequence of the virus was obtained by high-throughput sequencing. Combined with 34 monkeypox virus sequences downloaded from the NCBI and GISAID databases, a phylogenetic evolutionary tree was constructed using vaccinia, variola, and cowpox sequences as out groups. Results The whole genome sequence of the virus was obtained by both library construction methods, named hMpxV/China/CQ-CQCDC-001/2022, which belongs to the monkeypox virus branch lib, located in the same branch as hMpxV/Germany/BE-ChVir28656/2022 belonging to the IIb B.1 branch. Metagenomic libraries have more uniform sequence coverage than amplicon libraries, but the sequencing depth is lower and the effective data volume accounts for less. Conclusion Clade IIb B.1 monkeypox viruses have already been introduced into the mainland China, and the sequencing of subsequently discovered monkeypox viruses should be based on the sample size and sequencing timeframe, sequencing cost-effectiveness ratio, and other factors to choose the appropriate library construction method. [ABSTRACT FROM AUTHOR]

Objective To study the drug resistance phenotype, drug resistance gene, and virulence gene of Helicobacter pylori (Hp) in patients with failed eradication therapy, and to provide reference for remedial therapy. Methods Hp was isolated and cultured from gastric mucosa of patients with failed eradication therapy. Drug sensitivity test was used to detect the resistance of isolated strains to Metronidazole (MTZ), Clarithromycin (CLA), Levofloxacin (LEV), Amoxicillin (AMX), Tetracycline (TET), and Furazolidone (FZ). CLA and LEV resistance genes 23S rRNA, gyrA and virulence genes cagA, vacA, iceA, oipA were amplified by PCR, to further explore the relationship between drug resistance phenotype and drug resistance and virulence genes. Results In total 40 strains of Hp were isolated from 58 patients (69.0%). The resistance rates to MTZ, CLA, LEV, AMX, TET, and FZ were 100.0%, 82.5%, 72.5%, 0%, 0%, and 0%, respectively. The most common mutations of 23S rRNA and gyrA genes were T2182C (78.6%) and N87K (32.0%), respectively, and new C2165T and A2219G mutations were found in CLA resistant strains. In addition, the positive rates of virulence genes cagA, vac A si, vacA s2, vacA ml, vacA m2, tceAl, tceA2, and oipA were 97.1%, 100.0%, 0%, 42.9%, 57.1%, 62.9%, 11.4%, and 68.6%, respectively, and the difference was statistically significant. Conclusion The resistance of Hp isolates to MTZ, CLA and LEV in patients with failed eradication therapy in this area is serious, and AMX, TET and FZ are recommended for remedial therapy. The relationship between CLA resistance and C2165T and A2219G mutation and LEV resistance and vacA m2 warrants further investigation. [ABSTRACT FROM AUTHOR]

Abstract: Objective: This study is to examine the secretion effects of -galactosidase in Lactococcus lactis. Methods: The usp45 and β-galactosidase genes were cloned and inserted into plasmid pMG36e to obtain the recombinant plasmid pMG36e-usp-lacZ. This recombinant plasmid was transformed into both Escherichia coli DH5α and L. lactis MG1363. The enzyme activity, gene sequencing, SDS-PAGE and hereditary stability were assessed and studied. Results: The lacZ gene inserted into plasmids pMG36e-usp-lacZ was 99.37% similar to the GenBank sequence, and SDS-PAGE revealed an evident idio-strap at 116 KDa between L. lactis MG1363/pMG36e-usp-lacZ in both supernatant and cell samples. β-Galactosidase activity measured 0.225 U/mL in L. lactis pMG36e-usp-lacZ transformants, and its secretion rate was 10%. The plasmid pMG36e-usp-lacZ appeared more stable in MG1363. Conclusion: The authors concluded that these new recombinant bacteria well expressed and secreted β-galactosidase, indicating that the β-galactosidase expression system was successfully constructed, and this might provide a new solution for management of lactose intolerance specifically and promote the use of gene-modified organisms as part of the food-grade plasmid in general. [Copyright &y& Elsevier]

Objective: To investigate the status of infections caused by respiratory pathogens and the patterns of infections caused by pathogens in different seasons, age groups and stages of pneumoconiosis so as to explore the pathogen spectrum of respiratory tract infections in pneumoconiosis patients., Methods: The sputum samples of 376 pneumoconiosis patients admitted to an occupational disease hospital in Chengdu between January, 2017 and October, 2019 were collected. Clinical information of the patients was collected and lab tests were conducted to check for 23 kinds of common respiratory viruses, bacteria and fungi in the sputum. The relationship between seasons, ages, and different stages of pneumoconiosis and the pathogen detection rate was analyzed., Results: In the 376 sputum samples, the detection rates of pathogens, viruses, bacteria and fungi were 42.29% (159/376), 32.98% (124/376), 9.57% (36/376) and 6.12% (23/376), respectively. The six pathogens with the highest detection rates were parainfluenza virus, rhinovirus, influenza virus, Candida albicans , Klebsiella pneumoniae and Candida krousei . The severity of respiratory tract infection did not show significant difference in different seasons, age groups, and pneumoconiosis stages., Conclusion: The pathogen spectrum of respiratory tract infections in patients with pneumoconiosis is complicated and the proportion of viral infection is high. However, the severity of the infection is not associated with age, seasonal, or pneumoconiosis staging differences., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Sciences).)

Objective: To select and identify the bacterium which highly produces protease and β-D-glucosidase from 72 strains of Shuidouchi from Sichuan, and to provide evidence for further research on its nutritional value and fermentation strain exploiting., Methods: Casein degradation test and pNPG chemical test were applied respectively to detect the capacity to produce protease and β-D-glucosidase of each strain. Characteristics of morphology, biochemistry, 16S rRNA and MALDI-TOF-MS were used to identify the fermentation strain, which genetic stability, curves of growth and enzyme producing were also obtained., Results: The strain with the highest enzyme activity of β-D-glucosidase (0.084 U/L) among the top 10 strains for producing protease was selected as the fermentation strain and was identified as Bacillus subtilis , which curves of growth and enzyme producing conformed as well. The result of genetic stability showed that capacity of enzyme producing was stable until the 10th generation., Conclusions: The fermentation strain which highly produced protease and β-D-glucosidase was selected from 72 strains of shuidouchi from Sichuan and was identified as Bacillus subtilis ., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Science Edition).)

Objective: To investigate the prevalence and gene characteristics of different groups of human parainfluenza virus (HPIV) infection in hospitalized adults with acute respiratory tract infections (ARI)., Methods: RT-PCR was used to detect HPIV hemagglutinin (HA) DNA,which was extracted from sputum samples of 1 039 adult patients with ARI from March,2014 to June,2016. The HA gene amplified from randomly selected positive samples were sequenced to analyze the homology and variation., Results: 10.6% (110/1 039) of these samples were positive for HPIV,including 8 cases of HPIV-1,22 cases of HPIV-2,46 cases of HPIV-3 and 34 cases of HPIV-4. Detectable rate varied among different groups of HPIV according to seasons of the year and ages of patients. No significant differences were found between the positive samples and the reference sequences. Compared with different reference strains of different regions,the genetic distance of nucleotide is the smallest between the strains tested in this study and the reference strains of other provinces and cities in China., Conclusion: In Chengdu region,HPIV virus is highly detected in ARI,all subtypes were detected with HPIV-3 being the main subtype.

Aim: To investigate the anti-obesity and antibacterial effects of Ligustrum robustum (L. robustum) in vivo and in vitro and its possible mechanisms., Methods: The effects of L. robustum aqueous extract (LR) on various gut bacteria in vitro were evaluated. The effects of LR on high-fat diet-fed (HFD) rats in vivo were also assessed. Culture methods, quantitative polymerase chain reaction, and terminal-restriction fragment length polymorphism were used to analyze the effects of LR on gut bacteria. Biochemical tests were also performed to detect the changes in obesity-related indicators after LR treatment., Results: LR treatment lowered adipose weight and decreased Lee's index, blood glucose, total cholesterol, and lipid in the tested groups relative to control (P < 0.05). To determine the reasons for these changes, we assessed the potential bacteriostatic and bactericidal effects of LR on specific bacterial species in vitro. LR affected the richness, diversity, and evenness of gut bacteria, increased fecal Lactobacillus, and decreased Enterococci in HFD rats (P < 0.05)., Conclusion: L. robustum may be a safe and effective food for weight loss and obesity control, and the effects of L. robustum might be mediated by the regulation of gut bacteria.

Objective: To isolate aflatoxin-producing strains from paprika samples and to do a preliminarily study on the relationship between aflatoxin-producing ability and the genes aflR, omt-1 and ver-1., Methods: Fungi were isolated by traditional culture method. Potential aflatoxin-producing strains were screened by phenotypic traits and multiplex PCR. After these potential aflatoxin-producing strains cultured in the toxigenic culture medium, the levels of aflatoxin B, (AFB1) of the cultures were tested with ELISA method. The phylogenetic tree of aflR, omt-1 and ver-1 was constructed to explore the relationship between these genes and the AFB1-producing capacity., Results: 17 potential aflatoxin-producing fungi were isolated. The ratio of positive toxigenic strains is 64. 71%. 11 isolates were positive in AFB1 detection while existing high sequence homology with AS 3. 4408, 6 isolates were negative in AFB1 detection while existing high sequence homology with Aspergillus oryzae., Conclusion: Aspergillus flavus are potential candidates for aflatoxin control. Not all Aspergillus flavus have AFB1-producing capacity, aflR gene had a direct relation to AFB1-producing capacity, while ver-1 and omt-1 were related to the level of AFB1 producing.

Objective: To preliminary study of the resistance mechanisms of S. pneumoniae (S. pn) by determining the resistance rates and gene of S. pn isolated from the lower respiratory tract infection infants., Methods: Drug susceptibility test with disk diffusion and broth micro-dilution was conducted to evaluate the resistance rates of 73 strains of S. pn isolated from the lower respiratory tract infection infants to penicillin, levofloxacin and other 10 antibiotics. PCR method was used to analysis the antimicrobial resistant genes tet M, mef A, erm A, erm B and int Tn of the isolates., Results: The antibiotic resistance rates of the S. pn isolates to erythromycin, clindamycin and tetracycline were 95. 9%, 94. 5%, 87. 7% and 0% to vancomycin when tested with disk diffusion method. The antibiotic resistance rates of these isolates to penicillin, cefotaxime and ceftriaxone were 45. 2%, 47. 9% and 46. 6% respectively when tested with broth micro-dilution method. The carrier frequencies of tet M, mef A, erm A, erm B, int Tn genes in the 73 isolates were 91. 8%, 63. 0%, 58. 9%, 39. 7% and 61. 6% respectively., Conclusion: The S. pn strains isolated from infant respiratory tract in Chengdu perform a serious drug resistance problem, especially to routine antibiotics like erythromycin, clindamycin and tetracycline and cephalosporin, the resistance rate to levofloxacin, chloramphenicol remained at a low level; the resistance to tetracycline was closely related with the tet M gene fragment, the resistance to macrolide was mainly decided by active efflux pump and secondarily by the alternation of gene targeting, int Tn had close relation with tet M, erm B.

Objective: To establish a method for detecting 3 common toxigenic molds (Aspergillus, Penicillium, and Fusarium) based on non-modified magnetic beads coupled with multiple real-time PCR (NMB-multiple qPCR)., Methods: The primers and genus-specific probes were designed based on the rDNA sequences to develop a multiple real-time PCR using non-modified magnetic bead to enrichment of fungal spores. The sensitivity, specificity and repeatability of this assay were evaluated., Results: The detection limit of this assay for spiked samples was 10(4) CFU/g, demonstrating a 10-fold greater detection sensitivity of this assay than that of real-time PCR. The NMB-multiple qPCR assay also showed good specificity and reproducibility and yielded comparable results with those by traditional colony counting method for spiked samples (P>0.05)., Conclusion: NMB-multiple qPCR assay we established allows rapid and sensitive detection of common mycotoxigenic fungi in paprika.

Objective: To understand the variation of G glycoprotein gene of human respiratory syncytial virus (HRSV) obtained from Sichuan in 2010 and determine the dominant genotypes., Methods: G glycoprotein gene of seven cases of subtype A and eleven cases of subtype B of HRSV were amplified by RT-PCR and sequenced. The phylogenetic trees were constructed to determine the subtype of samples. And then, the genetic variations of the second hypervariable region of G glycoprotein gene were studied., Results: The nucleotide genetic distances of G glycoprotein gene in subtype A and subtype B HRSV were 0.022 +/- 0.005 and 0.073 +/- 0.010, respectively. Transitions were more prevalent than transversions, GA -AG were the most frequent transitions detected among group A viruses, while UC+CU transitions were the most among group B. Phylogenetic analyses demonstrated that 7 subtype A virus could be clustered into one genotype, genotype GA2, and 11 subtype B virus could be clustered into two genotypes, GB2 and BA. The length of G protein gene in group A was all 298aa, but in group B included 295aa, 312aa and 315aa. Selective pressure was purifying selection in both subtypes. 9 positively selected sites in group A and 1 in group B on the second hypervariable region of G protein were identified., Conclusion: GA2, GB2 and BA were the main genotype. The changes may favor virus escape from the host immune response including the variation of the G protein gene length, frequency of nucleotide changes and selective pressure.

Objective: To investigate the epidemiological features and clinical features of human bocavirus (HBoV) infection in children with respiratory tract infection in Sichuan, and to analysis the HBoV VP1 gene mutation characteristics of Sichuan clinical strains., Methods: Nasopharyngeal secretions were collected from 787 hospitalized children with respiratory tract infection. PCR was used to detect HBoV. The VP1 genetic variations of the nucleotide and amino acid were analysised respectively., Results: Out of 787 specimens from respiratory tract, 8.26% (65/787) were positive for HBoV, 50.77% (33/65) were co-detected with other respiratory viruses. HBoV is usually detected in children under 3 years of age, the positive rate of male children was higher than female children. Most frequently clinical symptoms of HBoV were cough, fever and expectoration. Phylogenetic analyses showed that all the 8 clinical strains were HBoV1 genetype. GA+AG transitions were the most frequent transitions detected, while the nonsynonymous mutations were more than synonymous mutations., Conclusion: HBoV is an important pathogen of respiratory tract infection in children in Sichuan. The main type of nucleotide variation is transitions. Amino acids mutations may relate to immune evasion.

Objective: To investigate the variation of cytotoxic T-lymphocyte (CTL) and neutralizing epitopes in F protein of respiratory syncytial virus (RSV) isolated in Sichuan., Methods: Nearly full-length of F protein gene of 10 strains of RSV isolated in Sichuan was amplified by RT-PCR and sequenced. The genetic variations, especially the CTL and neutralizing antibody epitopes within different subtypes and genotypes were analyzed and compared., Results: The F protein of RSV is highly conserved within the two subtypes, with the P-distances of nucleotide and amino acids were 0.102 +/- 0.005 and 0.058 +/- 0.006, respectively. Neutralizing epitopes 47F and L4 were conserved between the subtypes, but RS-348 and 7C2 were only conserved within the subtypes. CTL epitopes HLA B * 57, HLA A * 01 and HLA Cw * 12 were conserved only within subtype A. There were specific different sites between the subtypes., Conclusion: The sequences of F protein from Sichuan RSV isolates were highly conserved, so as the epitopes on F-protein within subtypes, the identified CLT epitopes in subtype A may not be recognized in subtype B virus.

Objective: To investigate the molecular mechanisms of reduced carbapenem susceptibility in Klebsiella pneumonia., Methods: One reduced carbapenem susceptible Klebsiella pneumonia clinical isolate was investigated. Kirby-Bauer disc test was applied to determine the antibiotic susceptibility of the isolate. Modified Hodge Test and EDTA-disk synergy test were used to confirm whether this Klebsiella pneumonia strain could produce metallo-beta-lactamase. The genotype of the beta-lactamase was confirmed by PCR and DNA sequence analysis. Plasmid DNA preparations and conjugation experiment were used to determine the location of the resistant gene., Results: Antibacterial circle of imipenem, meropenem for Klebsiella pneumonia isolate were 16 cm and 17 cm implied that the isolated strain producing carbapenemas. Modified Hodge Test and EDTA-disk synergy test confirmed that this Klebsiella pneumonia isolate produced metallo-beta-lactamase. IMP-4 gene was amplified by PCR and confirmed with sequence analysis. A reduced carbapenem susceptibility in obtained conjugants was observed when evaluated with Kirby-Bauer disc test and conjugation experiment also revealed that blalMP-4 were carried on one plasmid with a size of approximately 73 000 bp., Conclusion: Production of plasmid-mediated metallo-beta lactamase IMP-4 might lead to the reduced susceptibility of Klebsiella pneumonia spp. to carbapenems.

Objective: This study is to examine the secretion effects of beta-galactosidase in Lactococcus lactis., Methods: The usp45 and beta-galactosidase genes were cloned and inserted into plasmid pMG36e to obtain the recombinant plasmid pMG36e-usp-lacZ. This recombinant plasmid was transformed into both Escherichia coli DH5alpha and L. lactis MG1363. The enzyme activity, gene sequencing, SDS-PAGE and hereditary stability were assessed and studied., Results: The lacZ gene inserted into plasmids pMG36e-usp-lacZ was 99.37% similar to the GenBank sequence, and SDS-PAGE revealed an evident idio-strap at 116 KDa between L. lactis MG1363/pMG36e-usp-lacZ in both supernatant and cell samples. Beta-Galactosidase activity measured 0.225 U/mL in L. lactis pMG36e-usp-lacZ transformants, and its secretion rate was 10%. The plasmid pMG36e-usp-lacZ appeared more stable in MG1363., Conclusion: The authors concluded that these new recombinant bacteria well expressed and secreted beta-galactosidase, indicating that the beta-galactosidase expression system was successfully constructed, and this might provide a new solution for management of lactose intolerance specifically and promote the use of gene-modified organisms as part of the food-grade plasmid in general.

Objective: To study the genotype of rotavirus and the genetic variations of the major neutralization antigen VP4 of group A rotavirus in fecal samples from infants with diarrhea in Chengdu, Sichuan province, China., Methods: The fecal specimens were collected from infant patients with diarrhea in the spring of 2010 at West China Second University Hospital. Reverse transcription polymerase chain reaction (RT-PCR) was performed to identify rotavirus G serotypes and P genotypes. VP4 gene fragments of the virus were amplified from two strains drawn randomly from the prevailing genotype and cloned into a T-A clone vector to generate the recombinants for sequencing., Results: A group rotaviruses were detected in 13 of 75 specimens (17.3%). Serotype G1 was the predominant type (7/13) and two were serotype G3, four strains' serotypes were unidentified. Analysis of P gene demonstrated that genotype P [8] was the predominant type (6/13), whereas only two P[4] genotype were detected and genotypes for two strains were not determined. G1P [8] was the predominant type of G/P dominance combination (5/13). Sequencing results of the VP4 gene for the analyzed two strains implied that they were genotype P[8] with a 97% homology in sequence. Compared with the standard strain, homologies were also more than 90%., Conclusion: Rotavirus is one of the major etiological agents of viral diarrhea among infants in Chengdu. G1 was the dominant type G in Chengdu. G1P[8] was the predominant type of G/P dominance combination.

Objective: To explore the possibility of Bacteroides spp. as fecal contamination indicator bacteria with real-time quantitative PCR (RT-PCR) assay through analyzing the correlation between Bacteroides spp. and coliform group in external environment., Methods: Quantity of coliform group and Bacteroides in water samples were detected by most-probable-number method (MPN) and RT-PCR, respectively, and their detection correlation was evaluated with linear correlation analysis. Both methods were also applied to detect the contaminated time limits and river water samples collected at four sampling sites in three different times., Results: Seventy two hours were needed for the numeration of coliform group with MPN method, while RT-PCR could detect Bacteroides within 3 hours. The contaminated time limit of indoor and outdoor water samples of coliform group was more than 40 days and 9 days, and Bacteroides 13 days and 5 days, respectively. Also, the positive correlation between the quantity of Bacteroides and coliform group in outdoor water samples was obtained, the quantity of Bacteroides was from 8.3 × 10(6) copies/ml to less than 10(4) copies/ml during the first day to the fifth day, while coliform group was 4.3 × 10(6) MPN/100 ml to 2.4 × 10(3) MPN/100 ml. A 100% coincidence rate of the detection results with both methods was also observed. These results indicated that the detection results of both methods had perfect consistency., Conclusion: Bacteroides spp. can be potentially used as fecal contamination indicator bacteria with RT-PCR rapid detection.

Aim: To investigate the differences in cultivable gut bacteria and peroxisome proliferator-activated receptor γ2 (PPAR-γ2) gene Pro12Ala variation in obese and normal-weight Chinese people., Methods: Using culture methods, the amounts of Escherichia coli, Enterococci, Bacteroides, Lactobacilli, Bifidobacteria and Clostridium perfringens (C. perfringens) in the feces of 52 obese participants [body mass index (BMI): ≥ 28 kg/m(2)] and 52 participants of normal-weight (BMI: 18.5-24 kg/m(2)) were obtained. Study participants completed comprehensive questionnaires and underwent clinical laboratory tests. The polymerase chain reaction-restriction fragment length polymorphism (PCR-PFLP) assay was used to analyze PPAR-γ2 gene Pro12Ala variation., Results: The obese group exhibited a lower amount of C. perfringens (6.54 ± 0.65 vs 6.94 ± 0.57, P = 0.001) and Bacteroides (9.81 ± 0.58 vs 10.06 ± 0.39, P = 0.012) than their normal-weight counterparts. No major differences were observed in Pro12Ala genotype distribution between the two groups; however, obese individuals with a Pro/Ala genotype had a significantly lower level of Bacteroides (9.45 ± 0.62 vs 9.93 ± 0.51, P = 0.027) than those with a Pro/Pro genotype. In addition, the obese group demonstrated a higher stool frequency (U = 975, P < 0.001) and a looser stool (U = 1062, P = 0.015) than the normal-weight group., Conclusion: Our results indicated interactions among cultivable gut flora, host genetic factors and obese phenotype and this might be helpful for obesity prevention.

Objective: To study on the polymorphism of UCP2 gene in Chengdu simple obesity and normal-weight people and to initially investigate the relationship between UCP2 Ala55Val variation and gut bacteria., Methods: PCR-PFLP was applied to determine the genotypes of Ala55Val variant in the UCP2 gene of 86 Chengdu people (the simple obesity group, 43 subjects; the normal-weight group, 43 subjects). And six kinds of gut bacteria among different genotypes in different groups were analyzed., Results: Both the simple obesity and the normal-weight group had the Ala55Val variants of Ala/Ala, Val/Val and Ala/Val in the UCP2 gene, and the Ala55Val genotype distributions between the two groups was significantly different (chi2=11.97, P< 0.05). The allelic mutation frequency in the simple obesity group was higher than that of the normal-weight group (chi2=10.06, P<0.05). However, no significant difference was observed in the population of six gut bacteria among different genotypes in different groups (P>0.05)., Conclusion: The UCP2 gene mutation might be a risk factor of obesity in Chengdu area. However, this gene mutation may not be an impact factor on the alternation of gut bacteria.

Objective: To construct recombinant Lactococcus lactis strains exhibiting high beta-galactosidase activity in non-fusion way, and study their enzyme activities and enzyme secretion rates., Methods: The recombinant plasmids pMG36e-lacZ 1.1480 and pMG36e-lacZ wch9901 which could express beta-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus in non-fusion way in Escherichia coli were obtained and transformed into Lactococcus lactis subsp. lactis MG1363. The beta-galactosidase activity of resulting recombinant L. lactis in different incubation periods and lactose concentrations, and their enzyme secretion rates in different culture conditions were examined., Results: Recombinant L. lactis carrying pMG36e-lacZ wch9901 (MG1363/pMG36e-lacZ wch9901) exhibited the highest beta-galactosidase activity. Its enzyme activity was (16.95 +/- 0.09) U/mg pro, which was 2.75 folds of that of the native counterpart; recombinant L. lactis reached its enzyme producing peak after grown for 24 h; decreased enzyme activity of recombinant L. lactis were observed when incubated in medium containing lactose; the beta-galactosidase expressed by recombinant strains could be secreted into the culture medium, and the highest secretion rate (27.09 +/- 0.05)% was observed when the culture medium contained 20 g/L of lactose and without erythromycin., Conclusion: High level expression of non-fusion beta-galactosidase with secretion in recombinant L. lactis strains was obtained. This will be very helpful for the further developing of live delivery bacteria of beta-galactosidase.

Objective: To construct four recombinant Lactococcus lactis strains exhibiting high beta-galactosidase activity in fusion or non-fusion ways, and to study the influence factors for their protein expression and secretion., Methods: The gene fragments encoding beta-galactosidase from two strains of Lactobacillus bulgaricus, wch9901 isolated from yogurt and 1.1480 purchased from the Chinese Academy of Sciences, were amplified and inserted into lactococcal expression vector pMG36e. For fusion expression, the open reading frame of the beta-galactosidase gene was amplified, while for non-fusion expression, the open reading frame of the beta-galactosidase gene was amplified with its native Shine-Dalgarno sequence upstream. The start codon of the beta-galactosidase gene partially overlapped with the stop codon of vector origin open reading frame. Then, the recombinant plasmids were transformed into Escherichia coli DH5 alpha and Lactococcus lactis subsp. lactis MG1363 and confirmed by determining beta-galactosidase activities., Results: The non-fusion expression plasmids showed a significantly higher beta-galactosidase activity in transformed strains than the fusion expression plasmids. The highest enzyme activity was observed in Lactococcus lactis transformed with the non-fusion expression plasmids which were inserted into the beta-galactosidase gene from Lactobacillus bulgaricus wch9901. The beta-galactosidase activity was 2.75 times as high as that of the native counterpart. In addition, beta-galactosidase expressed by recombinant plasmids in Lactococcus lactis could be secreted into the culture medium. The highest secretion rate (27.1%) was observed when the culture medium contained 20 g/L of lactose., Conclusion: Different properties of the native bacteria may have some effects on the protein expression of recombinant plasmids. Non-fusion expression shows a higher enzyme activity in host bacteria. There may be a host-related weak secretion signal peptide gene within the structure gene of Lb. bulgaricus beta-galactosidase, and its translation product may introduce the enzyme secretion out of cells in special hosts.

Objective: To construct the food-grade recombinant probiotic strain with high activity beta-galactosidase, the beta-galactosidase gene (lacZ)from Lactobacillus delbrueckii subsp. bulgaricus was in non-fusion expressed in Escherichia coli., Methods: From Lb. delbrueckii subsp. bulgaricus lacZ gene, the DNA sequence containing Shine-Dalgarno (SD) and ATGA sequences between upstream 18 bp and downstream 1 bp at start codon ATG was selected as upstream primer for PCR amplifying lacZ gene. Then lacZ cDNA was inserted into expression plasmid pMG36e to construct recombinant expression plasmid. Recombinant plasmids were introduced into E. coli, and positive clones were screened. To identify the gene recombination, the recombinant plasmid was cut by restriction enzyme and sequenced. To identify the protein expression, the beta-galactosidase activities of recombinant strains were determined., Results: The restriction maps of recombinant plasmids were acceptable. The gene inserted into the recombinant plasmid had more than 99% homology with the lacZ gene of Lb. delbrueckii subsp. bulgaricus. The enzyme activity and enzyme activity ratio of E. coli DH5 alpha carrying pMG36e-lacZ 1.1480 were 3.074 U/mL and 6.939 U/mg pro respectively. The enzyme activity and enzyme activity ratio of E. coli DH5a carrying pMG36e-lacZ wch9901 were 4.755 U/mL and 8.537 U/mg pro respectively., Conclusion: lacZ from Lb. delbrueckii subsp. bulgaricus have gotten non-fusion expression in E. coli. The SD and ATGA sequences we selected can introduce lacZ non-fusion expression in E. coli.

Objective: To screen bacteria for the engineering bacteria expressing and secreting high activity beta-galactosidase, and two bacterial strains called as R92-2 and R111 with acid and bile resistances would be isolated from health human intestine to strain identification and phylogenetic analysis., Methods: These two strains were first been identified with phenotype characteristic analysis. Then the 16S rDNAs of these two bacterial strains were amplified and sequenced with the primers designed by the conserve sequences. DNA sequences were blasted against GenBank, and the phylogenetic trees were constructed., Results: Phenotype characteristic analysis showed that both of the bacterial strains belonged to lactic acid bacteria. Results of Blastn showed that 16S rDNAs of R92-2 and Weissella cibaria had 100% homology; 16S rDNAs of R111 and Enterococcus faecium had 100% homology too., Conclusion: R92-2 belongs to Weissella cibaria strain; R111 belongs to Enterococcus faecium strain. These two stains may be used to construct the engineering bacteria expressing and secreting high activity beta-galactosidase.

Objective: To identify the relationship between amino acid mutations in Neisseria gonorrhoeae isolates and their antibiotic resistance., Methods: PI gene fragments of Neisseria gonorrhoeae from 17 clinical isolates were obtained with PCR amplification. They were cloned into the PCR cloning vector pBS-T to form pBS-T-PI and sequenced. The sequences of PI genes were analyzed. At the same time, minimum inhibitory concentration (MIC) of penicillin and tetracycline to these 17 isolates were measured and contrasted with the corresponding PI sequence., Results: The recombinants of PI gene from 17 clinical isolates of Neisseria gonorrhoeae were successfully constructed and sequenced. They were divided into PIA and PIB subtypes according to the results from blastn software by comparing the sequences with the GenBank. Mutations were found at the sites of 120 and 121. There were only some of the sequences having an aspartic acid (D) mutation on 120 and 121 sites, which was not the same as reported. On the other hand,there were two PI sequences,5-9 and 6-1, whose mutations on No. 120 were lysine, similar to those documented., Conclusion: Some relationship between PI amino acids mutations at sites 120 and 121 in Neisseria gonorrhoeae isolates from Chengdu, China and their resistance to penicillin and tetracycline were found. However,further studies need to be done in the future to confirm this hypothesis.

Objective: To express recombinant Salmonella invasion protein A(InvA) in E. coli and purify it., Methods: The invA gene of Salmonella was amplified by PCR from the Salmonella genome and cloned into expression vector pET-30c (+) to generate the pET-invA recombinants. They were comfirmed by restriction endonuclease digestion and sequence analysis. The verified recombinant was transformed into E. coli BL21 (DE3). After inducing with isopropyl-beta-D-thiogalactopyranoside (IPTG), the recombinant protein was purified via Ni-NTA affinity chromatography under denature conditions. The recombinant proteins were analyzed with SDS-PAGE and Western blot., Results: The pET-invA recombinant for Salmonella was successfully constructed and the recombinant InvA protein was expressed in E. coli at a relatively high level. The SDS-PAGE results for the purified recombinant protein demonstrated that the purified protein had reached the electrophoresis purity., Conclusion: The successful expression of the Salmonella InvA protein will be very helpful for the further study on its antigenicity, immunological activity, and the development of rapid detection methods for Salmonella strains.

Objective: To construct neisseria surface protein (NspA) recombinants of Neisseria gonorrhoeae from a reference strain and express this protein in E. coli., Methods: The fragments of NspA gene of Neisseria gonorrhoeae was amplified by PCR from the reference strain genomic DNA and cloned into expression vector pET-30c (+) to get the pET-NspA recombinants. The recombinants were verified with restrictive endonuclease digestion and sequence analysis. The verified recombinant was transformed into E. coli BL21 (DE3). After inducing with IPTG, the expressed NspA protein was analyzed by SDS-PAGE and Western Blot., Results: The pET-NspA expression recombinants for the reference strain of Neisseria gonorrhoeae were successfully constructed and the induced recombinant NspA protein was observed., Conclusion: The successful expression of the Neisseria gonorrhoeae NspA protein will be very helpful for the further research of its antigenicity and immunological activity, and for the construction of preventive vaccines on Neisseria gonorrhoeae infection.

Objective: To investigate the alteration of dominating intestinal floras among groups of people with different body fat and probe into the possible effect on lipid metabolism., Methods: According to the BMI values, subjects were divided into 4 groups, including fleshless group, normal group, overweight group and obese group. Five dominating floras of all fresh stools were quantitated using selective culture method, and all data were analyzed statistically., Results: With the increase of BMI values, there is a decreasing trend in the amount of Lactobacillus, Bifidobacterium and Enterobacillus, and the amounts of Bacteriodes obviously increased (P < 0.01). No obvious alternation of the amounts of Enterococcus and Clostridium was observed., Conclusion: Bacteriodes may exert an boosting effect on the pile of fat and development of obese, however Enterobacillus, Lactobacillus, and Bifidobacterium have a potential opposite effect.

Objective: To construct expressing recombinant of Mn-SOD of Deinococcus radiodurans and express the target protein in E. coli BL21(DE3)., Methods: SOD gene was amplified by PCR from genomic DNA of Deinococcus radiodurans and inserted into expression plasmid pET-30a(+) to create the recombinant pET-SOD. After being analyzed by the restriction endonuclease, the plasmid was transformed into E. coli BL21(DE3), and the recombinant protein was expressed after induction by the isopropyl-beta-D-thiogalactopyranoside (IPTG) and was analyzed with SDS-PAGE., Results: The recombinant plasmid pET-SOD was obtained, and the recombinant protein was highly expressed in E. coli BL21(DE3). The activity of recombinant superoxide dismutase was 51,800 U per gram of wet bacteria., Conclusion: This study has provided a foundation for further studies and applications of the recombinant Mn-SOD.

Objective: To construct Neisseria gonorrhoeae major outer membrane protein PI gene recombinants for expression of the target protein in E. coli., Methods: Four clinic isolates of Neisseria gonorrhoeae were collected, and then the genome DNA of these strains was extracted. The gene encoding for PI of Neisseria gonorrhoeae was amplified by PCR, inserted into the cloning vector pBS-T; the recombinant plasmids pBS-T-PI-NG were constructed and sequence analysis was performed. Then PI gene fragments were inserted into expression vector pET30b to form pET30b-PI-NG recombinants. The PI protein expression was induced by adding IPTG in the inocula. The expressed proteins were analyzed by SDS-PAGE., Results: The pET30b-PI-NG expression recombinants for four clinic isolates of Neisseria gonorrhoeae were constructed successfully; three of the four expression recombinants were expressed successfully., Conclusion: The expressed PI protein will be applied in the further research for PI antigenicity and immunological activity. This will be very helpful for the further construction of preventive vaccines directed against Neisseria gonorrhoeae infection.

Objective: To develop a rapid detection method for genetically modified soybean resistant to glyphosate., Methods: A duplex PCR was performed with primers designed in this study to simultaneously amplify heterogenous genes in transgenic soybean: CaMV-35S promoter, NOS terminator and CP4-EPSPS gene. And a simple capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was developed and applied to the rapid analysis of the above PCR products by using a 50 cm length x 100 microm i.d. capillary coated with linear polyacrylamide and an 8 g/L HPMC-4000 sieving buffer under 200 V/cm electric field strength., Results: The proposed method was able to simultaneously detect the three heterogenous genes existing in genetically modified soybean under the optimization conditions of PCR and capillary electrophoresis. The measured sequences of the duplex PCR products were identical with the original genes' sequences. Moreover, the sample volumes required were not more than 5 nl and the detection could be completed in less than 24 min. The relative standard deviations (R. S. D.) of the migration times for the PCR products were < or = 3.2%., Conclusion: In comparison with agarose gels electrophoresis, the duplex PCR-based capillary electrophoretic method with laser-induced fluorescence detection is rapid, sensitive and accurate, and it is suitable for detection of genetically modified soybean.

Objective: To obtain the gene which encode high activity beta-galactosidase from Lactobacillus bulgaricus and study the influencing factors of amplification., Methods: Lysozyme and freeze-thaw cycles were applied to obtain Lactobacillus bulgaricus DNA; different template concentration and annealing temperature were selected for the amplification., Results: 60 ng/microliter template concentration and 66 degrees C annealing temperature were the best reaction conditions of amplifying the gene., Conclusion: This study optimized the template concentration and anneal temperature conditions of amplifying Lactobacillus bulgaricus beta-galactosidase gene, thus providing a foundation for further studies.