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The aim of this study was to develop a semi-quantitative immunochromatographic method for rapid detection of Newcastle disease virus (NDV) antibodies by expressing HN protein in rice endosperm bioreactor. The recombinant plasmid pUC57-HN was digested by MlyⅠ and XhoⅠ to retrieve the HN gene, while the intermediate vector pMP3 containing promoter, signal peptide and terminator was digested by NaeⅠ and XhoⅠ. The HN gene and the linearized pMP3 were purified and ligated to form a recombinant plasmid pMP3-HN1. Subsequently, pMP3-HN1 and plant vector pCAMBIA1300 were digested by EcoRⅠ and Hind Ⅲ, and the HN1 gene was cloned into pCAMBIA1300. The recombinant plasmid pCAMBIA1300-HN1 was introduced into Agrobacterium tumefaciens EHA105 by electrotransformation, and the pCAMBIA1300-HN1 was transferred into rice callus by agrobacterium-mediated method. After dark culture, callus screening, differentiation, rooting and transplanting, transgenic rice seeds were obtained 4 months later. PCR identified that the HN gene has been inserted into the rice genome. SDS-PAGE and Western blotting indicated that the HN protein was successfully expressed in the positive rice endosperm. The purity of the HN protein was more than 90% by SP cation exchange chromatography and gel filtration chromatography. According to the national standards for the diagnostic techniques of Newcastle disease HI test (HI≥4log2, positive antibody reaction), a colloidal gold labeled purified HN protein was used to prepare a semi-quantitative test strip by double-antibody sandwich method for rapid detection of NDV antibody. The results showed that the test strip did not cross-react with positive sera against other viruses, and the sensitivity of the test strip reached 1:102 400 for standard positive sera of Newcastle disease. Testing of a total of 308 clinical sera showed that the compliance rate of the test strip with HI test was 97.08%, and the Kappa value was 0.942. In conclusion, high purity recombinant HN protein was obtained from rice endosperm, and a simple, rapid, highly sensitive and highly specific semi-quantitative immunochromatographic strip was developed. The test strip could be used for immune evaluation of the Newcastle disease vaccine.
Subject(s)
Animals , Antibodies, Viral , Chickens , HN Protein/metabolism , Newcastle Disease/prevention & control , Newcastle disease virus/metabolism , Oryza/geneticsABSTRACT
Objective To understand the prevalence of drug resistance in treatment-naive injecting drug users (IDUs) infected with HIV-1 in Guangzhou.Methods HIV-1 RNA were extracted from the serum specimens of the newly confirmed HIV-1 positive IDUs living in Guangzhou,being infected through injecting drug use and receiving no antiretroviral therapy at the time of confirmation during 2008-2015.Full sequence of pol protease (PR) gene and partial sequence of reverse transcriptase (RT) gene were amplified by nested reverse transcription polymerase chain reaction (nested-PCR) and sequenced.After that,data were submitted to the HIV resistance database of Stanford University for drug resistance analysis.Results Among the 518 HIV-1 infected IDUs,H1V-1 pol gene segments were successfully obtained from the serum samples of 407 HIV-1 infected IDUs (78.57％) aged 18-64 (37.44 ± 8.14) years.Among them,males accounted for 89.68％ (365/407),those of Han ethnic group accounted for 89.93％ (366/407),the unmarried accounted for 55.28％ (225/407),and those with education level of junior high school or below accounted for 83.78％ (341/407).The distribution of subtypes was predominated by CRF07_BC (47.18％,192/407),followed by CRF01_AE (23.83％,97/407),CRF08_BC (22.85％,93/407),and other subtypes (6.14％,25/407).The overall prevalence of drug resistance was 3.44％ (14/407).The prevalence of drug resistance to protease inhibitors,nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors were 1.47％(6/407),0.25％ (1/407) and 1.72％ (7/407) respectively.The mutation rate was 12.29％ (50/407).No major drug resistance mutation was detected in protease and nucleoside reverse transcriptase regions.Higher rate of V179E mutation in the non-nucleoside reverse transcriptase region was detected in other subtypes and subtype CRF07_BC.Mutation seemed to have occurred in all 8 cases of subtype CRF55_01B in other subtypes.The highest mutation rate of E138A was detected in subtype CRF08_BC (3.23％).Two cases were resistant to all four drugs of NNRTIs.Conclusions The prevalence of drug resistance in treatment-naive HIV-1 positive IDUs remained at a relatively low level during 2008-2015,in Guangzhou.Most infections were sensitive to existing antiviral drugs.However,drug resistance surveillance in IDUs infected with HIV should be strengthened to prevent the prevalence of multi-drug resistance and cross drug resistance.
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Objective To understand the prevalence of drug resistance in treatment-naive injecting drug users (IDUs) infected with HIV-1 in Guangzhou.Methods HIV-1 RNA were extracted from the serum specimens of the newly confirmed HIV-1 positive IDUs living in Guangzhou,being infected through injecting drug use and receiving no antiretroviral therapy at the time of confirmation during 2008-2015.Full sequence of pol protease (PR) gene and partial sequence of reverse transcriptase (RT) gene were amplified by nested reverse transcription polymerase chain reaction (nested-PCR) and sequenced.After that,data were submitted to the HIV resistance database of Stanford University for drug resistance analysis.Results Among the 518 HIV-1 infected IDUs,H1V-1 pol gene segments were successfully obtained from the serum samples of 407 HIV-1 infected IDUs (78.57％) aged 18-64 (37.44 ± 8.14) years.Among them,males accounted for 89.68％ (365/407),those of Han ethnic group accounted for 89.93％ (366/407),the unmarried accounted for 55.28％ (225/407),and those with education level of junior high school or below accounted for 83.78％ (341/407).The distribution of subtypes was predominated by CRF07_BC (47.18％,192/407),followed by CRF01_AE (23.83％,97/407),CRF08_BC (22.85％,93/407),and other subtypes (6.14％,25/407).The overall prevalence of drug resistance was 3.44％ (14/407).The prevalence of drug resistance to protease inhibitors,nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors were 1.47％(6/407),0.25％ (1/407) and 1.72％ (7/407) respectively.The mutation rate was 12.29％ (50/407).No major drug resistance mutation was detected in protease and nucleoside reverse transcriptase regions.Higher rate of V179E mutation in the non-nucleoside reverse transcriptase region was detected in other subtypes and subtype CRF07_BC.Mutation seemed to have occurred in all 8 cases of subtype CRF55_01B in other subtypes.The highest mutation rate of E138A was detected in subtype CRF08_BC (3.23％).Two cases were resistant to all four drugs of NNRTIs.Conclusions The prevalence of drug resistance in treatment-naive HIV-1 positive IDUs remained at a relatively low level during 2008-2015,in Guangzhou.Most infections were sensitive to existing antiviral drugs.However,drug resistance surveillance in IDUs infected with HIV should be strengthened to prevent the prevalence of multi-drug resistance and cross drug resistance.
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Abnormal modification in protein Neddylation is closely related to the occurrence and progression of various tumors. Studies have shown that the level of enzymes participating in Neddylation is higher than that in normal neighboring tissues,so the relationship between Neddylation and cancer has aroused much concern.Ned-dylation has been considered to be a novel anticancer target. Neddylation inactivation by a first-in-class small molecular inhibitor,MLN4924,induces cell cycle arrest,apoptosis,senescence and autophagy in different tumor cells.MLN4924 also exerts significant anticancer effects by inhibiting tumor angiogenesis and regulating the func-tion of immune cells. In this review,the latest progress of protein Neddylation and tumor cell cycle,apoptosis, senescence,autophagy,angiogenesis and regulation of immunocyte were briefly introduced to provide theoretical reference for the development of antitumor drugs targeting Neddylation.
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Objective To evaluate the role of transient receptor potential channel 8 (TRPM8) in the development and maintenance of neuropathic pain and the relationship with nuclear factor kappa B (NF-κB) in the dorsal root ganglion of rats.Methods Thirty-six pathogen-free healthy adult male SpragueDawley rats,weighing 180-200 g,aged 6-8 weeks,were divided into 3 groups (n=12 each) using a random number table:sham opeation group (group S),neuropathic pain group (group NP) and TRPM8 bocker BCTC group (group BCTC).Neuropathic pain was induced by ligation of the right sciatic nerve in anesthetized rats.BCTC 20 nmol was intrathecally injected once a day on preoperative day 1 and postoperative days 1,3,7 and 10 in group BCTC.The thermal,mechanical and cold pain thresholds were measured on preoperative day 1 and postoperative days 1,3,7,10 and 14.The dorsal root ganglions of the lumbar segment (L4-6) were removed on postoperative days 7 and 14 for determination of the expression of TRPM8 and NF-κB p65 by Western blot.Results Compared with group S,the thermal and mechanical thresholds were significantly decreased on postoperative days 1-14,and the cold pain threshold was decreased on postoperative days 3-14 in NP and BCTC groups,the expression of TRPM8 and NF-κB p65 in dorsal root ganglions was up-regulated on postoperative days 7 and 14 in group NP (P＜0.05),and no significant change was found in the expression of TRPM8 and NF-κB p65 in dorsal root ganglions in group BCTC (P＞0.05).Compared with group NP,the thermal pain threshold was significantly decreased and the cold pain threshold was increased on postoperative days 3-14,the expression of TRPM8 and NF-κB p65 in dorsal root ganglions was up-regulated on postoperative days 7 and 14,and no significant change was found in the mechanical pain threshold in group BCTC (P＞0.05).Conclusion NF-κB activation after opening of TR-PM8 in dorsal root ganglion neurons is involved in the development and maintenance of neuropathie pain in rats.
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Objective To evaluate the role of P2X3 receptors in the development and mnaintenance of neuropathic pain (NP) and the relationship with nuclear factor kappa B (NF-κB) in dorsal root ganglia of rats.Methods Thirty-six SPF healthy male Sprague-Dawley rats,weighing 180-200 g,aged 4-6 weeks,in which intrathecal catheters were successfully implanted,were divided into 3 groups (n =12 each) using a random number table:sham operation group (group S),NP group and P2X3 receptor antagonist A-317491 group (group A).At 3 days after intrathecal catheters were successfully implanted,NP was induced by chronic constriction injury to the sciatic nerve.The sciatic nerve was only exposed but not occluded in group S.Intrathecal injection was performed twice a day for 14 consecutive days starting from 1 day after operation.Normal saline 20 μ l was intrathecally injected in group S and group NP,and A-317491 100 nmol/10 μ1 and normal saline 10 μ l were intrathecally injected in group A.Thermal paw withdrawal threshold (TWT) and mechanical paw withdrawal threshold (MWT) were measured on day 1 before operation and at 30 min after intrathecal injection on days 1,3,7,10 and 14 after operation.On days 7 and 14 after operation,the rats were sacrificed and the dorsal root ganglia of the lumbar segment were removed for determination of the expression of P2X3 receptors and NF-κB p65 by Western blot.Results Compared with group S,TWT and MWT were significantly decreased at each time point after operation,and thc expression of P2X3 receptors andi NF-κB p65 in dorsal root ganglia was up-regulated on days 7 and [4 after operation in group NP (P＜0.05 or 0.01).Compared with group NP,TWT was significantly increased at each time point after operation,MWT was increased on days 3,7,10 and 14 after operation,anl the expression of P2X3 receptors and NF-κB p65 in dorsal root ganglia was down-regulated on days 7 and 14 after operation in group A (P＜0.05).Conclusion The mechanism underlying the development and maintenance of NP is related to the up-regulation of P2X3 receptor expression and promotion of the expression of NF-κB in dorsal root ganglia of rats.
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AIM:To investigate the effects of Qili Qiangxin capsule on serum adiponectin ( APN) , serum N-terminal pro-brain natriuretic peptide ( NT-proBNP) and heart function in the patients of coronary heart disease combined with congestive heart failure .METHODS: One hundred and twenty patients were randomly divided into treatment group and control group , and both groups were given anti-failure routine therapy .The patients in treatment group were treated with Qili Qiangxin capsule and the patients in control group were treated with placebo .The patients in the 2 groups were given a certain dose of the drugs for 6 months.The New York Heart Association (NYHA) heart function classification, left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD),left ventricular ejection frac-tion (LVEF), and 6-min walking test (6-MWT) were observed before and after treatment .The levels of APN, NT-proBNP were measured by ELISA before and after treatment .RESULTS:With the increase in the class of NYHA heart function , the serum concentrations of APN and NT-proBNP in the heart failure cases increased significantly .After 6-month treat-ment, the effective rate in experimental group was 91.7%and that in control group was 75.0%.A significant difference was found between the 2 groups (P<0.01).After treatment, LVEDD and LVESD in both groups were decreased signifi-cantly, and LVEF in both groups was increased significantly .The serum concentrations of APN and NT-proBNP decreased significantly (P<0.05).6-MWT result was improved significantly.Compared with control group, more obvious effect was observed in experimental group ( P<0.05) .CONCLUSION:Treatment with Qili Qiangxin capsule reduces the levels of APN and NT-proBNP in the patients with coronary heart disease combined with congestive heart failure .
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Objective To investigate the effects of the zinc finger protein A20 on LPS-TLR4 signaling pathways in severe acute pancreatitis(SAP) rats.Methods 24 SD rats were randomly divided into 3 groups:group A(the sham operation group),group B (the SAP group),group C (the SAP group treated with LPS).SAP model was induced by retro-injection of intraductal 5％ sodium taurocholate into the biliary-pancreatic duct as previously described.The protein expression of A20,TLR4,NF-κBp65 and p38MAPK in pancreatic tissues was evaluated by immunohistochemistry.Results The positive area of A20 in pancreatic tissues was decreased in group B and group C compared with that in group A (t =17.234,19.698,all P ＜ 0.05).On the contrary,the expression of TLR4,NF-κBp65 and p38MAPK in pancreatic tissues were up-regulated(t =15.909,20.432,16.543,18.629,22.105,19.006,all P ＜ 0.05).A20 was decreased in group C than that in group B (t =14.894,P ＜ 0.05),while TLR4、NF-κBp65 and p38MAPK were increased in group C than those in group B (t =14.047,15.582,17.070,all P ＜0.05).Conclusion The expression of A20 reduced and TLR4,NF-κBp65 and p38MAPK enhanced in the pancreas of rats with SAP,which indicated that A20 inhibited LPS-TLR4 signaling pathways which play important roles in the pathogenesis of SAP.
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Objective To investigate of the tissue TGF-β changes at early stage of hypertrophic scar formation and the value of scar blisters in hypertrophic scar.Methods The TGF-β1 content in the blister fluid and the blood were quantified with ELISA,patients(n=15)with hypertrophy scar after depth burn were included,three time point(each n=5)on early stage(<3 months)of hypertrophy scar formationwere monitored.and normal skin blister fluid and the blood(n=5)was used as control.Results The serum TGF-β1 in the both hypertrophic scar patients and normal skin group was not elevated(P>0.01),the TGF-β1 in the blister of normal skin was also not elevated(P>0.01),but TGF-β1 level in the scarblisters hypertrophic scar was elevated significantly[<60 d(158.5±69.8)pg/L,60-90 d,(181.1±40.1)pg/L,>90 d,(534.4±125.9)pg/L,P<0.01] and higher than the normal skin blister and the blood(P<15.6 pg/L.P<0.01),the increased TGF-β1 1evel in the hypertrophic scar blisters were persisted for at least three months.the TGF-β1 level of scar blister on the 3th month of hypertrophic scar formation reached a peak [(534.4±125.9)pg/L,P<0.01].Conclusions The data in this study indicates that TGF-β production at the early stage of hypertrophic scar formation is increased and may play an important role in scar formation;scar blisters is a valuable approach in hypertrophic scar study.
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The erythrocyte complement receptor 1 (ECR1) activity and CR1 quantitative genotype distribution were studied and the mechanisms of decreased ECR1 activity in cerebral infarction revealed. By using red blood cell yeast rosette test ECR1 activities were measured and by using PCR-RFLP CR1 Hind Ⅲ genomic polymorphism detected in the patients with cerebral infarction and healthy controls. The results showed that the level of C3bRR was decreased and the level of CICRR increased in the patients with cerebral infarction as compared with healthy controls (both P<0.05). CR1 quantitative genotype distribution in the patients with cerebral infarction was differed significantly from that of healthy controls (P<0.05). It was concluded that the decrease of ECR1 activity in the patients with cerebral infarction was correlated with CR1 Hind Ⅲ genomic polymorphism.
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Objective To investigate the effect of Ganoderma Triterpene(GT)on the expression of CD69 and HLA-DR on human T lymphocyte.Methods The activation of human T lymphocytes was detected and analyzed by flow cytometry after co-culturing with different concentrations of GT for 6 hours or for 72 hours.Results GT stimulated the expression of CD69 on T lymphocytes,the effect being significant at GT concentration of 10 ? g/mL(P
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Objective To evaluate HRCT typing of solitary pulmonary nodule with resected.Methods 30 cases with solitary plumonary nodules (totally 41) were undergone CT and HRCT scan befor operation.HRCT appearances were retrospectively analyzed and categorized into two types: typeⅠ ground-glass opacity (pure ground-glass opacity, mixed lower opacity, central high opacity with ground opacity ), typeⅡ pure solid opacity.Results TypeⅠ:14 nodules included bronchioloalveolar cell carcinoma (9 nodules) , adenocarcinoma (3 nodules),inflammatory granuloma (1 nodule) and adenoma (1 nodule).TypeⅡ : 27 nodules included adenocarcinoma(13 nodules),bronchioloalveolar cell carcinoma(4 nodules), squamous cell carcinoma(3 nodules),small cell lung cancer(1 nodule),adenoma(2 nodules),tuberculosis(2 nodules), inflammatory granuloma(1 nodule) and hamartoma(1 nodule). Among nodules with resected, ground-glass opacity was found more frequently in bronchioloalveolar cell carcinoma than in non- bronchioloalveolar cell carcinoma (P=0.002).No significant difference in ground-glass opacity existed between tumor lesions and non-tumor lesions (P=0.282) or adenocarcinoma and non-adenocarcinoma (P=0.146).Conclusion In the findings of HRCT in solitary pulmonary nodules,the nodules of ground-glass opacity find more frequently in bronchioloalveolar cell carcinoma than that in other lesions.
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AIM:To investigate the affecting factors of detecting platelet activation by flow cytometry (FCM). METHODS:Using decoagulant of natrium citricum,anticoagnlated peripheral venous bloods from 6 healthy donors were labeled with the method of three-colour immunofluorescence assay. Platelet activation markers fibrinogen receptor (Fib-R,PAC-1) and P-selectin (CD62P) were measured. In the same time,the reproducibility of FCM was assessed.RESULTS:The platelet activation markers PAC-1 and CD62P at each time point showed significant difference(P