Pushing the Limits of Capacitively Coupled Contactless Conductivity Detection for Capillary Electrophoresis.

Capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D) has proven to be an efficient technique for the separation and detection of charged inorganic, organic, and biochemical analytes. It offers several advantages, including cost-effectiveness, nanoliter injection volume, short analysis time, good separation efficiency, suitability for miniaturization, and portability. However, the routine determination of common inorganic cations (NH4+, K+, Na+, Ca2+, Mg2+, and Li+) and inorganic anions (F-, Cl-, Br-, NO2-, NO3-, PO43-, and SO42-) in water quality monitoring typically exhibits limits of detection of about 0.3-1 µM without preconcentration. This sensitivity often proves insufficient for the applications of CE-C4D in trace analysis situations. Here, we explore methods to push the detection limits of CE-C4D through a comprehensive consideration of signal and noise sources. In particular, we (i) studied the model of C4D and its guiding roles in C4D and CE-C4D, (ii) optimized the bandwidth and noise performance of the current-to-voltage (I-V) converter, and (iii) reduced the noise level due to the strong background signal of the background electrolyte by adaptive differential detection. We characterized the system with Li+; the 3-fold signal-to-noise (S/N) detection limit for Li+ was determined at 20 nM, with a linear range spanning from 60 nM to 1.6 mM. Moreover, the optimized CE-C4D method was applied to the analysis of common mixed inorganic cations (K+, Na+, Ca2+, Mg2+, and Li+), anions (F-, Cl-, Br-, NO2-, NO3-, PO43-, and SO42-), toxic halides (BrO3-) and heavy metal ions (Pb2+, Cd2+, Cr3+, Co2+, Ni2+, Zn2+, and Cu2+) at trace concentrations of 200 nM. All electropherograms showed good S/N ratios, thus proving its applicability and accuracy. Our results have shown that the developed CE-C4D method is feasible for trace ion analysis in water quality control.

Calibration of the oscillation amplitude of quartz tuning fork-based force sensors with astigmatic displacement microscopy.

Quartz tuning forks and qPlus-based force sensors offer an alternative approach to silicon cantilevers for investigating tip-sample interactions in scanning probe microscopy. The high-quality factor (Q) and stiffness of these sensors prevent the tip from jumping to the contact, even at sub-nanometer amplitude. The qPlus configuration enables simultaneous scanning tunneling microscopy and atomic force microscopy, achieving spatial resolution and spectroscopy at the subatomic level. However, to enable precise measurement of tip-sample interaction forces, confidence in these measurements is contingent upon the accurate calibration of the spring constant and oscillation amplitude of the sensor. Here, we have developed a method called astigmatic displacement microscopy with picometer sensitivity.

Effect of Different Modes of Administration of Dexmedetomidine Combined with Nerve Block on Postoperative Analgesia in Total Knee Arthroplasty.

INTRODUCTION: Dexmedetomidine (DEX) as a nerve block adjuvant can significantly prolong analgesia. However, whether perineural or systemic administration of DEX is more beneficial in patients undergoing total knee arthroplasty (TKA) has not been thoroughly investigated. To this end, we evaluated the effects of perineural and systemic DEX administration on postoperative analgesia in patients undergoing TKA surgery. METHODS: We randomly assigned patients undergoing TKA under general anesthesia combined with femoral nerve block and sciatic nerve block to one of three groups: (1) ropivacaine plus perineural dexmedetomidine (DP): 0.25% ropivacaine 40 mL plus 0.5 µg/kg dexmedetomidine; (2) ropivacaine plus systemic dexmedetomidine (DS): 0.25% ropivacaine 40 mL plus systemic 0.5 µg/kg dexmedetomidine; (3) control group (C): 0.25% ropivacaine 40 mL. RESULTS: The average length of time until patients first experienced postoperative pain was significantly longer in the DP group (26.0 h [22.0-30.0 h]) than in the DS group (22.4 h [18-26.8 h]) and the control group (22.9 h [19.5-26.3 h], P = 0.001). For this result there was no significant difference between the DS and the control group. Compared with the DS and control groups, patients in the DP group had lower resting visual analogue scale (VAS) scores at 24, 48, and 72 h after surgery (P < 0.05). VAS activity scores at 12, 24, and 48 h after surgery in the DP group were lower than those in the DS and control groups, with a statistically significant difference (P < 0.05). Compared with the DS and control groups, the amount of postoperative opioids in the DP group was also significantly reduced, and the number of people needing postoperative rescue analgesia was significantly lower, with a statistical difference (P < 0.05). Meanwhile, the sleep satisfaction of patients in the DP group on the first night after surgery and the satisfaction with pain control at 72 h after surgery were both higher than those in the DS group and control group (P < 0.05). CONCLUSIONS: Perineural administration of DEX can significantly prolong the interval until patients report pain for the first time after TKA, relieve postoperative pain, reduce postoperative opioid dosage, and improve postoperative sleep quality and satisfaction with pain control. TRIAL REGISTRATION: The trial was registered at the Chinese Clinical Trial Registry, identifier ChiCTR1900025808.

Effects of Different Local Analgesic Techniques on Postoperative Quality of Life and Pain in Patients Undergoing Total Hip Arthroplasty Under General Anesthesia: A Randomized Controlled Trial.

BACKGROUND: Both lumbosacral plexus block (LSPB) and local infiltration analgesia (LIA) can provide postoperative analgesia for patients undergoing total hip arthroplasty (THA). The current study aimed to compare the differences between LSPB and LIA on postoperative pain and quality of life (QoL) in THA patients. METHODS: A total of 117 patients aged 40-80 years, ASA I-III, were prospectively randomized into two groups: a general anesthesia plus LSPB (Group LSPB) and a general anesthesia plus LIA (Group LIA). Pain intensity and opioid consumption were recorded Within 72 hours after surgery. QoL was measured by EQ-5D and EQ-VAS questionnaires, and the incidence of postoperative pain was measured as part of the EQ-5D on day 1, day 3, day 7, and month 1, month 3, and month 6 after surgery. RESULTS: EQ-5D scores: Mobility, Self-Care, Usual Activities, Pain/Discomfort, and Anxiety/Depression were higher in Group LSPB versus Group LIA throughout six-month follow-ups (p = 0.039). The pain intensity was lower in Group LSPB than in Group LIA 0-12 h after surgery (2.41 vs 2.79, p = 0.01), but was higher in Group LSPB than in Group LIA 12-24 h (2.59 vs 2.05, p = 0.02) and 24-48 h (2.18 vs 1.73, p = 0.02) after surgery. There were no differences in opioid consumption between the groups during the first 72 postoperative hours. In the first month after surgery, more patients in Group LSPB than in Group LIA had no pain (52 vs 40, p = 0.04). CONCLUSION: Both LSPB and LIA can provide satisfactory postoperative analgesia. The LSPB is better than LIA for long-term QoL in THA patients undergoing general anesthesia. CLINICAL TRIAL REGISTRATION NUMBER: The Chinese Clinical Trial Registry (ChiCTR-INR-17012545).

Chemical constituents from common vetch (Vicia sativa L.) and their antioxidant and cytotoxic activities.

Vicia sativa L. (common vetch) is a potential food source for both human beings and animals because of its abundant nutritional composition. There is a lack of phytochemical study on the whole plant, and thus the objective of this study was to investigate the isolation of phytochemicals and evaluate their biological activities. A new flavanol, (2R,3S)-3,3'-dihydroxy-4',7-dimethoxyflavanol (1), together with nine known compounds, two flavones (2-3), one coumarin (4), and six oleanane triterpenoids (5-10), was obtained from Vicia sativa L.. The structure of the new compound 1 was determined via its NMR spectra, IR and CD data. Compound 3 displayed the potential of the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging effect in antioxidant test. In terms of cytotoxic activities, compound 3 showed moderate cytotoxic activities against three human tumor cells, especially HeLa cells.

Pelvic plexus block to provide better anesthesia in transperineal template-guided prostate biopsy: a randomised controlled trial.

BACKGROUND: To evaluate the efficacy of pelvic plexus block (PPB) in relief pain during transperineal template-guided prostate biopsy (TTPB), compared with conventional periprostatic nerve block (PNB). METHODS: From July 2016 to August 2017, 245 patients who were performed TTPB in Clinical Medical College of Yangzhou University were recruited. The patients were randomized into three groups using a random number table. Group-1 received prostate capsule local anesthesia with 22 ml of 1% lidocaine. Group-2 additionally received PNB on the basis of Group-1. To perform PNB, 5 ml 1% lidocaine was injected into the region of prostatic neurovascular bundle situated in the angle of prostate-bladder-seminal vesicle. Group-3 received prostate capsule local anesthesia plus PPB (5 ml of 1% lidocaine injection into the pelvic plexus which located on lateral to the bilateral seminal vesicle apex). The patients' pain and satisfaction were evaluated by visual analogue scale and visual numerical scale, respectively. RESULTS: The age, total prostate volume, PSA and the number of cores were comparable among the three groups. The visual analog scale scores of group-3 were significantly lower than group-2 during biopsy (P = 0.003). Conversely, the visual numeric scale scores were higher in group-3 (P = 0.039). Both the group-2 and group-3 outperformed the group-1 in alleviating pain and had a higher quantification of satisfaction. There were no significant differences in the pain scores or the satisfaction scores at 30 min after the procedure among the three groups. CONCLUSIONS: The analgesic efficacy of PPB guided by Doppler ultrasound in TTPB was better than that of PNB and both were superior to no nerve block. TRIAL REGISTRATION: ChiCTR-IOR-17013533 , 01/06/2016.

Permanent 125 I prostate brachytherapy for castration-resistant prostate cancer.

OBJECTIVE: To evaluate the clinical significance of permanent 125 I prostate brachytherapy in patients with castration-resistant prostate cancer. METHODS: A retrospective study of 45 patients with castration-resistant prostate cancer from the Clinical Medical College, Yangzhou University, Yangzhou, Jiangsu, China was carried out. Patients were divided into two groups according to different treatments: 21 patients received endocrine therapy alone (control group), and 24 patients underwent brachytherapy combined with endocrine therapy (treatment group). Prostate-specific antigen progression-free survival, cancer-specific survival, overall survival and quality of life of the two groups were compared. RESULTS: The median prostate-specific antigen progression-free survival and cancer-specific survival of the treatment group were 29 months (interquartile range 25-37 months) and 37 months (interquartile range 30-50 months), respectively. These were significantly longer than those of the control group (both P < 0.05). Prostate-specific antigen (before androgen deprivation therapy and before brachytherapy), prostate volume, Gleason score, clinical stage and brachytherapy were associated with prostate-specific antigen progression-free survival and cancer-specific survival on univariate analysis. For the quality of life after treatment, urinary symptoms/problems at 1 month after brachytherapy compared with the control group had a statistically significant difference and clinically relevant deterioration, but after 6 months there were no statistically significant differences and clinically relevant deterioration. Compared with the control group, the physical functioning, social functioning, global health and general physical discomfort of the treatment group were significantly improved. CONCLUSIONS: Brachytherapy with 125 I seed implantation can effectively prolong survival of patients with castration-resistant prostate cancer and, to a certain extent, improve patients' quality of life.

Analysis of kinetoplast cytochrome b gene of 16 Leishmania isolates from different foci of China: different species of Leishmania in China and their phylogenetic inference.

BACKGROUND: Leishmania species belong to the family Trypanosomatidae and cause leishmaniasis, a geographically widespread disease that infects humans and other vertebrates. This disease remains endemic in China. Due to the large geographic area and complex ecological environment, the taxonomic position and phylogenetic relationship of Chinese Leishmania isolates remain uncertain. A recent internal transcribed spacer 1 and cytochrome oxidase II phylogeny of Chinese Leishmania isolates has challenged some aspects of their traditional taxonomy as well as cladistics hypotheses of their phylogeny. The current study was designed to provide further disease background and sequence analysis. METHODS: We systematically analyzed 50 cytochrome b (cyt b) gene sequences of 19 isolates (16 from China, 3 from other countries) sequenced after polymerase chain reaction (PCR) using a special primer for cyt b as well as 31 sequences downloaded from GenBank. After alignment, the data were analyzed using the maximum parsimony, Bayesian and netwok methods. RESULTS: Sequences of six haplotypes representing 10 Chinese isolates formed a monophyletic group and clustered with Leishmania tarentolae. The isolates GS1, GS7, XJ771 of this study from China clustered with other isolates of Leishmania donovani complex. The isolate JS1 was a sister to Leishmania tropica, which represented an L. tropica complex instead of clustering with L. donovani complex or with the other 10 Chinese isolates. The isolates KXG-2 and GS-GER20 formed a monophyletic group with Leishmania turanica from central Asia. In the different phylogenetic trees, all of the Chinese isolates occurred in at least four groups regardless of geographic distribution. CONCLUSIONS: The undescribed Leishmania species of China, which are clearly causative agents of canine leishmaniasis and human visceral leishmaniasis and are related to Sauroleishmania, may have evolved from a common ancestral parasite that came from the Americas and may have split off earlier than the other old world Leishmania. Our results also suggest the following: the isolates GS7, GS1 and XJ771 occur as part of the L. donovani complex; the JS1 isolate is L. tropica; and the isolate GS-GER20 identified as Leishmania gerbilli is close to KXG-2 which is L. turanica.

Phylogenic analysis of Chinese Leishmania isolates based on small subunit ribosomal RNA (SSU rRNA) and 7 spliced leader RNA (7SL RNA).

The leishmaniases are zoonotic diseases caused by protozoan parasites of the genus Leishmania. Leishmaniases are still endemic in China, especially in the west and northwest froniter regions. To revalue the preliminary phylogenetic results of Chinese Leishmania isolates, we amplified partial fragment of small subunit ribosomal RNA (SSU rRNA) and 7 spliced leader RNA (7SL RNA), then tested the phylogenetic relationships among Chinese Leishmania isolates and their relatives by analyzing SSU rRNA gene sequences and 7SL RNA gene sequences. 19 SSU RNA sequences and 9 7SL RNA sequences were obtained in our study, then analyzed with 42 SSU RNA sequences and 32 7SL RNA sequences retrieved from Genbank, respectively. In the Bayesian analysis of the SSU RNA gene, the isolate MHOM/CN/93/GS7 and the isolate IPHL/CN/77/XJ771 are members of Leishmania donovani complex, while the isolate MHOM/CN/84/JS1 clustered with Leishmania tropica. The other 11 Chinese Leishmania isolates (MHOM/CN/90/WC, MCAN/CN/90/SC11, MHOM/CN/80/XJ801, MHOM/CN/85/GS4, MHOM/CN/84/SD1, MCAN/CN/86/SC7, MHOM/CN/54/#3, MHOM/CN/83/GS2, MHOM/CN/90/SC10H2, MHOM/CN/89/GS6 and MHOM/CN/ 89/GS5) form an unclassified group, defined as Leishmania sp., and the most relative species to this group is L. tarentolae. In the Bayesian analysis of the 7SL RNA gene, 9 Chinese Leishmania isolates also formed an unclassified group with L. tarentolae, including canine isolate 10, MHOM/CN/85/GS4, MHOM/CN/84/SD1, MCAN/CN/86/SC7, MHOM/CN/54/#3, MHOM/ CN/83/GS2, MHOM/CN/90/SC10H2, MHOM/CN/89/GS6 and MHOM/CN/89/GS5. We concluded that: (1) Chinese Leishmania isolates are non-monophyly group; (2) an unclassified group may exist in China, and the most relative species to this group is L. tarentolae; (3) MHOM/CN/84/JS1, which was previously assigned as L. donovani, was most genetically related to L. tropica strain MHOM/SU/74/K27.

Protective immunity against Legionnaires' disease in an A/J mouse model using a DNA vaccine composed of an outer membrane protein (29 kDa) and the pilE fusion protein.

In order to assess the protective effects of the DNA vaccine (pcDip/pilE) against Legionella pneumophila, the coding sequences of the 2 proteins were cloned into the pET32a(+) and pcDNA3.1(+) vectors. To provide an enhanced immunological response, the proteins were linked together. In this study, the A/J mouse model was used for examine the immunogenicity and protective efficacy of the DNA vaccines of pcDip, pcDpilE and pcDip/pilE. Our results showed that the total IgG titers were higher level increasing after the stimulation of pcDip/pilE than pcDip and pcDpilE immunization. The DNA vaccine (pcDip/pilE) can protect the A/J mouse against a higher dose (2 × 10(7)L. pneumophila cells) of L. pneumonia compared to the other single-DNA vaccine in our study, and the ratio of the survival reached 100% in 10 days after the last DNA vaccine immunization. Our study indicates that these findings provide experimental evidence to support the claim that pcDip/pilE may be an efficient DNA vaccine against Legionella pneumophila.

[Molecular identification, phylogenic and virulence analysis of a Legionella strain isolated from aquatic environment].

OBJECTIVE: CD-1 was isolated from aquatic environment, which grew with strict L-cysteine dependence. In this study, we applied molecular methods to identify CD-1, and animal test to understand its virulence. METHODS: To identify CD-1 strain, CD-1 strain was tested for genus-specific 16S rRNA of Legionella via PCR amplification, then its rpoB gene was sequenced for phylogenic analysis. To understand the virulence, CD-1 was detected for mip gene, which was an indispensable virulent gene of Legionella. Then, BABL/c mice were infected by CD-1 in different dosages. RESULTS: For identification, CD-1 was positive for genus-specific 16S rRNA of Legionella, while in the phylogenic tree CD-1 was a sister to Legionella longbeachae with high posterior probability (PP = 1.00). For the virulence analysis, CD-1 was positive for mip gene detection. In the animal test, all mice tested died when the infection dose of CD-1 strain reached 10(7) cfu/mL. CONCLUSION: CD-1 strain was identified to be Legionella longbeachae with strong virulence to BALB/c mice. It may be a potential virulent strain to human. This is the first strain of Legionella longbeachae isolated in Sichuan province, and this is the first virulence analysis of Legionella strain isolated from aquatic environment in China.

Application of multilocus sequence analysis (MLSA) for accurate identification of Legionella spp. Isolated from municipal fountains in Chengdu, China, based on 16S rRNA, mip, and rpoB genes.

Legionellosis (Legionnaires' disease; LD) is a form of severe pneumonia caused by species of Legionella bacteria. Because inhalation of Legionella-contaminated aerosol is considered the major infection route, routine assessments of potential infection sources such as hot water systems, air-conditioner cooling water, and municipal fountains are of great importance. In this study, we utilized in vitro culture and multilocus sequence analysis (MLSA) targeting 16S rRNA, mip, rpoB, and mip-rpoB concatenation to isolate and identify Legionella spp. from 5 municipal fountains in Chengdu City, Sichuan Province, China. Our results demonstrated that 16S rRNA was useful for initial identification, as it could recognize isolates robustly at the genus level, while the genes mip, rpoB, and mip-rpoB concatenation could confidently discriminate Legionella species. Notably, the three subspecies of L. pneumophila could be distinguished by the analysis based on rpoB. The serotyping result of strain CD-1 was consistent with genetic analysis based on the concatenation of mip and rpoB. Despite regular maintenance and sanitizing methods, 4 of the 5 municipal fountains investigated in this study were positive for Legionella contamination. Thus, regularly scheduled monitoring of municipal fountains is urgently needed as well as vigilant disinfection. Although the application of MLSA for inspection of potential sites of infection in public areas is not standard procedure, further investigations may prove its usefulness.

Serodiagnosis efficacy and immunogenicity of the fusion protein of Mycobacterium tuberculosis composed of the 10-kilodalton culture filtrate protein, ESAT-6, and the extracellular domain fragment of PPE68.

In order to identify immunodominant antigens of Mycobacterium tuberculosis that may be used in the serodiagnosis of active tuberculosis (TB), we designed an M. tuberculosis fusion protein consisting of CFP-10 (10-kDa culture filtrate protein), ESAT-6 (6-kDa early secreted antigenic target), and the extracellular domain fragment of PPE68 (PPE68'). Then, the coding sequences of the three proteins were inserted into a prokaryotic expression vector, pET-32a(+). To enhance the immunological response, the proteins were linked together. The fusion proteins with a 6 × His tag were successfully overexpressed in Escherichia coli BL21 and purified. The purified proteins were applied for detection of the total IgG titer by using an enzyme-linked immunosorbent assay (ELISA) with human sera from well-characterized TB cases and the control cases, and results were compared to those with purified protein derivative tuberculin (PPD). The ELISA results showed that among 140 cases of confirmed active TB and 70 control cases, CFP-10-ESAT-6-PPE68' had a sensitivity of 73.3% and specificity of 94.3%, compared to a sensitivity of 66.7% and specificity of 74.3% for PPD and a sensitivity of 65% and specificity of 91.4% for CFP-10-ESAT-6. In addition, the fusion protein CFP-10-ESAT-6-PPE68' stimulated a higher level of antigen-specific gamma interferon (IFN-γ) release for active-TB patients than PPD and CFP-10-ESAT-6. After immunization of C57BL/6 mice, the findings indicated that the total IgG titers and the concentrations of IFN-γ in mice immunized by CFP-10-ESAT-6-PPE68' were high and induced strong, long-term humoral immunity compared to results with PPD and CFP-10-ESAT-6. Thus, our study indicates that the fusion protein CFP-10-ESAT-6-PPE68' may be useful as an immunodominant antigen for the serodiagnosis of active TB.

Evaluation of the protective immunity of the Legionella pneumophila recombinant protein FlaA/MompS/PilE in an A/J mouse model.

To investigate the protect effects of the recombinant protein FlaA/MompS/PilE against Legionella pneumophila (L. pneumophila), the coding sequences of the three proteins were optimized by DNA Star software firstly, cloned, expressed by Escherichia coli BL21, and purified. To give an enhanced the immunological response, the proteins were linked together with (Linker) or without a linker insert (NLinker) and were purified from E. coli BL21. The A/J mouse model was used to determine the level of the induction of protective immunity from the purified proteins. Our results showed that the IgG titer, which was measured by ELISA, was increased after the administration of the five proteins. Compared to the administration of the individual proteins, the chimeric Linker and NLinker proteins displayed lasting immunity to a lethal dose of L. pneumophila challenge. The Linker protein protected the A/J mouse against a higher dose of L. pneumonia compared to the other proteins used in this study, as it contained a more effective immunogen. The work presented here demonstrates that the bioinformatics software, DNA Star, is a valid tool to analyse the epitopes of proteins and was useful in the optimization of proteins that could induce the protective immune response to L. pneumophila. The cross-immunity of recombinant proteins, such as the Linker and the NLinker chimera, have higher generates a greater immune than the single proteins.

Production and immunoanalytical application of 32 monoclonal antibodies against metacestode somatic antigens of Echinococcus multilocularis.

Alveolar echinococcosis is a rare but potentially fatal disease. Immunodiagnosis based on antibodies or antigens plays an important role in its diagnosis. In this study, metacestode somatic antigens of Echinococcus multilocularis were used to immunize BALB/c mice, and hybridomas were formed by cell fusion. Making use of the inherent effect of monoclonal antibody techniques to isolate different epitopes, we obtained a repertoire of 32 monoclonal antibodies against the metacestode somatic antigens. These monoclonal antibodies were used to investigate the specificity and localization of the metacestode antigens by enzyme-linked immunosorbent assay and immunohistochemistry, respectively. Nine antibodies specifically reacted with E. multilocularis, while 14 and ten cross-reacted with Echinococcus granulosus and Taenia saginata, respectively. Twenty-five antibodies stained the laminated layer. Eight reacted with the tegument of the protoscolex. Fourteen antibodies recognized the germinal layer. Most of the monoclonal antibodies can react with the antigen Em2. One antibody can react with antigen Em2 and Em10. One antibody that cross-reacted with T. saginata stained the germinal layer and protoscolex, especially its hooklets and suckers, but could not react with Em2 and Em10 antigens. It detected protein bands at 26 and 52 kDa. Two E. multilocularis-specific monoclonal antibodies stained both the germinal and laminated layers and could be used not only to purify specific antigens but also for immunohistochemical studies of E. multilocularis. In summary, these 32 monoclonal antibodies could have potential applications as useful tools in further studies of E. multilocularis antigen profiles.

[Expression of human TFF3 in Escherichia coli].

OBJECTIVE: To construct recombinant human TFF3 prokaryotic expressing plasmid, express it in E. coli and identify the expressed protein. METHODS: The cDNA for mature peptide of TFF3 was amplified by RT-PCR from RNA of human colon tissue and inserted into the MCS of the prokaryotic expressing plasmid pET32a (+). Then TFF3 was expressed as a fusion protein by IPTG induction. The recombinant protein was determined by SDS-PAGE and Western blot with a rabbit anti-TFF3 polyclonal antibody. RESULTS: Sequencing result indicated that the obtained TFF3 fragment was inserted into plasmid pET32a (+) successfully and the sequence was correct. The expression level of the fusion protein was highest after 6h induction with 1 mmol/L IPTG. The result of Western blot demonstrated that the relative molecular mass of recombinant protein was about 24 x 10(3) and the protein had good antigenicity and specificity. CONCLUSION: The expression plasmid pET32a-TFF3 was constructed and expressed successfully. This study will provide a substantial basis for further study of human TFF3.

[Construction of the eukaryotic expression plasmid containing lvgA gene flanked with CpG motifs of Legionella pneumophila and its expression in NIH3T3 cells].

OBJECTIVE: To construct the eukaryotic expression plasmid containing lvgA gene flanked with CpG motifs of Legionella pneumophila for its expression in NIH3T3 cells. METHODS: lvgA gene flanked with CpG motifs of Legionella pneumophila was amplified by PCR. The PCR products was inserted into the eukaryotic expression plasmid pcDNA3.1/myc-his(+) to construct the recombinant plasmid pclvgA/CpG, which was subsequently transfected into NIH3T3 cells via lipofection. Immunofluorescence analysis was carried out to detect the transient expression of the plasmid in the cells. RESULTS: Sequence analysis showed that the recombinant plasmid pclvgA/CpG contained the lvgA/CpG fragment with a length of 657 bp, encoding a protein of 27.7 Ku. Immunofluorescence analysis identified the transient expression of the recombinant plasmid pclvgA/CpG in NIH3T3 cells. CONCLUSION: The lvgA gene flanked with CpG motifs of Legionella pneumophila has been constructed successfully, and the transient expression of the recombinant plasmid pclvgA/CpG can be detected in NIH3T3 cells.

[Cloning and expression of extracellular region gene located in N-terminus of Leishmania Donovani].

The objective of this study was to construct and express recombinant prokaryotic plasmid pET32a (+)- ast1 in E. coli BL21(DE3). Amastin gene was amplified from genomic DNA of Leishmania Donovani and its transmembran region was predicted by the methods of SOSUI and Tmpred; astl located in N-terminus of amastin gene was amplified and cloned into prokaryotic plasmid pET32a(+), which was named pET32a(+)-ast1, and then rAST1 was expressed in E. coli BL21(DE3). The results of SDS-PAGE and immunobloting assay showed that a fusion protein rAST1 (relative molecular mass about 27 kDa) was able to express in BL21. The recombinant prokaryotic plasmid pET32a(+)- ast1 was successfully constructed, and noted to be efficiently expressed in E. coli BL21(DE3).

[Production of a monoclonal antibody specific to protoscolex of Echinococcus multilocularis].

OBJECTIVE: To prepare monoclonal antibody (McAb) specific to protoscolex of Echinococcus multilocularis. METHODS: BALB/c mice were immunized with crude antigen derived from E. multilocularis metacestodes. Spleen cells from immunized BALB/c mice were fused with SP2/0 myeloma cells by using hybridoma technique. ELISA and immunohistochemical staining were used to select hybridomas that secreted McAb P325 which especially against protoscolex. The number of metaphase chromosomes of hybridoma cells was counted. Characteristics of McAb P325 were identified by ELISA and immunohistochemical staining. RESULTS: One hybridoma cell clone secreting McAb against protoscolex was obtained. The number of metaphase chromosomes found in hybridoma cells was 98, which showed the characteristics of their parents. Immunohistochemical analysis showed that McAb P325 demonstrated binding activity to the germinal layer and protoscolex of E. multilocularis, especially to the hooklets and suckers, while did not bind with E. granulosus metacestodes and Cysticercus tenuicollis. CONCLUSION: The McAb is a valuable tool for immunohistochemical analysis, cell classification of E. multilocularis protoscolex, and study of specific antigen.