Objective: To fuse Mycobacterium tuberculosis protective antigen gene with mice ubiquitin gene, constructing antigen ubiquitin system. Methods: Mice ubiquitin cDNA was amplified by RT PCR from mice testicle,and 4 antigen genes were obtained by PCR from cultured Mycobacterium tuberculosis . Ubiquitin and 4 antigen genes were linked by flexible adaptor respectively and the fusing genes were cloned into pVAX vector.The recombinant plasmids were digested with endonuclease and sequenced.Then the recombinant plasmids were transfected into COS7 cells and the expression was assayed by ELISA. Results:Ubiquitin and 4 antigen genes were 0.2,0.3,0.7,1.0,1.65 kb in length by agarose electrophoresis. Endonuclease digestion of the recombinant plasmids indicated that the fusion genes were correctly inserted into pVAX vector. Sequencing results of fusion nucleic acid vaccines were identical to those in GenBank.The recombinant plasmids expressed in COS7 cells. Conclusion: Four Chinese Mycobacterium tuberculosis protective antigens ubiquitin systems are successfully constructed and can be expressed in eukaryotic cells. This may provide a basis for diagnosis and therapy of tuberculosis.

Neuropathic pain(NP)is directly caused by lesions or diseases of the somatosensory system.It can be attributed to a variety of causes and has a complex pathological mechanism.Purinergic P2X receptors play a critical role in NP.P2X receptors will be activated by the extracellular adenosine triphosphate(ATP)to mediate the pain when the tissue is damaged.In vertebrates,there are 7 subtypes of P2X receptors(P2X1-7)encoded by genes,among which the receptors of P2X3,P2X4,and P2X7 are closely related to NP.Acupuncture produces good therapeutic efficacy on NP.To explore the mechanism of purinergic signaling involved in acupuncture analgesia,a review of the actions of P2X3,P2X4,and P2X7 receptors in acupuncture for NP was conducted.The P2X3 receptor is specifically expressed in small-and moderate-sized neurons in the dorsal root ganglion of the primary afferent nerve and trigeminal ganglion.Acupuncture can directly mediate analgesia by down-regulating the expression of P2X3 receptor in neurons.P2X4 and P2X7 receptors mainly exist in the spinal microglia.Acupuncture can inhibit the nociceptive transmission of substantia gelatinosa neurons by regulating P2X4 receptor in spinal microglia.Moreover,acupuncture may alleviate NP by inhibiting the secretion of inflammatory factors by reducing P2X7 receptor in the spinal microglia.

OBJECTIVE:To prepare Carbinoxamine maleate sustained-release suspension,and evaluate its quality. METH-ODS:Using carbinoxamine maleate as raw material,drug-loaded resin was prepared by cation exchange resin;surface coating method was used to finally prepare sustained-release suspension,using Eudragit RS100 as sustained-release coating material to pre-pare sustained-release microparticles. HPLC was conducted to determine the content of carbinoxamine maleate,release degree of original preparations and self-made suspensions was compared,drug-loading capacity was calculated. RESULTS:The drug amount in preparing drug-loaded resin was 2%,reaction temperature was 25 ℃,and reaction time was 4 h;the drug-loading capacity in surface coating was 35%,amount of coating material was 10%,and reaction temperature was 40 ℃. The drug-loading capacities of sustained particles before and after coating were 35.23%,32.72%,respectively;the yield was 96.82%. The carbinoxamine ma-leate in prepared sustained-release suspension accounted for 98.76% of the labeled amount;release degree in 10 h reached about 80%,f2 was 65.73. CONCLUSIONS:Carbinoxamine maleate sustained-release suspension is prepared successfully,and its release is similar to the original preparation.

Objective: To clone porcine IL-4 cDNA and observe its adjuvanticity of vaccine against T. solium cysticerco-sis. Methods:The cDNA of porcine IL-4 was amplified by RT-PCR, which had 5' AUG initiatory codon with optimized trans-lational initiation. After sequencing,the cDNA was intergrated into expression vector pcDNA and transient expression was performed. Then newborn pigs were immunized with IL-4 expression vector and protective antigen DNA vaccine against T. solium cysticercosis. Results:The obtained sequence of porcine IL-4 cDNA was the same as reported. IL-4 and protective antigen could effectively promote the humoral response. Conclusion:An efficient expression plasmid containing porcine IL-4 cDNA is constructed. Its adjuvanticity of DNA vaccine against T. solium cysticercosis is preliminarily conformed.

Objective To explore the protective effect of pre-operation administration of nicorandil on myocardial injury and recurrent angina in patients with unstable angina pectoris who underwent percutaneous coronary intervention (PCI). Methods A total of 91 patients with unstable angina pectoris were admitted for PCI and randomly divided into 2 groups who either received nicorandil(5 mg tid)or not for 7 days prior to the procedure and routine dose of nicorandil (5mg tid) after it. The concentrations of creatine kinase MB (CK-MB) and cardiac troponin I(cTnI) were compared between two groups before PCI and 6 hours, 18 hours and 24 hours after PCI;Recurent angina and major adverse cardiac events,includ-ing death,re-infarction and stroke at 6 months after the procedure were also chased and compared. Results In total, 75 pa-tients who were successfully undergone elective PC1 were finally enrolled,among whom 37 cases were in nicorandil group and 38 cases were in routine group. Post-procedural levels of CK-MB and cTnI significantly reduced in the nicorandil group between 6~24 h (P<0.05) compared those in routine group.At 6 months follow-up,symptoms of recurrent angina after PCI were significantly relieved in the nicorandil group compared with that in routine group (P < 0.05).Conclusion nicorandil can limit the PCI-induced myocardial injury and relieve the symptoms of recurrent angina after PCI in patients with unstable angina.

Objective: To explore the effect of trimetazidine (TMZ) on T-peak (Tp) to T-end (Te) interval of resting ECG in patients with unstable angina pectoris (UAP) after percutaneous coronary intervention (PCI). Methods: We investigated 94 UAP patients with PCI and 76 of them ifnished the study as 2 groups. Control group,n=42, the patients received conventional treatment, and TMZ group,n=34, in addition to conventional treatment, the patients received TMZ 60 mg at 0.5 to 1 hour before PCI. The changes of Tp to Te interval before and after PCI were calculated, serum levels of creatine kinase (CK), creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) at before and at 6, 24 hours after PCI were compared between 2 groups. Ventricular arrhythmia was recorded during PCI. Results: Both groups had shortened Tp to Te intervals after PCI, and TMZ group had more shortened Tp to Te interval, bothP<0.05, while the Tp to Te intervals were similar between 2 group before PCI,P>0.05. The occurrence rate of ventricular arrhythmia in Control group was higher than that in TMZ group (12.52% vs 5.16%) during PCI,P<0.05. Compared with Control group, TMZ group had lower levels of CK, CK-MB and cTnI at 6, 24 hours after PCI, allP<0.05. Conclusion: TMZ could decrease Tp to Te interval of resting ECG, prevent ventricular arrhythmia and reduce the relevant myocardial injury in UAP patients after PCI.

Objective To analyse the immunostimulatory activity of CpG sequences in cysticercus cellulosae paramyosin (also named Antigen B,AgB)cDNA. Methods C57BL/6 mice were immunized with pcDNA3 AgB plasmid,pcDNA3 AgB′(CpG sequences were mutated),pcDNA3 or AgB protein and two weeks later,immune response was assayed by ELISA. Results IgG and IgG 2a were detectable at week 2 after immunization and continually increased until week 4.The antibody levels elicited by pcDNA3 AgB were significantly higher( P

Objective To clone and express the gene encoding ESAT antigen of Mycobacterium tuberculosis (chinese stain). Methods Genome of mycobacterium tuberculosis was extracted. Then, the full length cDNA encoding ESAT protein was amplified by PCR and cloned into prokaryotic expressing vector pGEX5T and sequenced. pGEX5T ESAT was expressed in k802 Ecoli, and the expressed protein was determined by western blot using the sera from ten patients with tuberculosis. Results One specific band of 0.3kb or so was obtained and sequencing result was identical to that reported from Genbank. The expressed protein could be specifically recognized by the sera from tuberculosis patients. Conclusions The full length cDNA of Mycobacterium tuberculosis (Chinese strain) ESAT protein was cloned and expressed successfully, which will be helpful in further studies on diagnosis and treatment of tuberculosis.

ObjectiveTo construct the eukaryotic coexpression plasmid CEA/IL-2, and to lay the foundation for further studying CEA nucleiotide vaccine , adjuvant and their effects of special antitumor immunity.MethodsThe eukaryotic expression plasmid (pcDNA3-CEA)containing the gene coding for CEA was obtained by RT-PCR and gene recombination techniques.Using enzymolysis,ligation and other techniques,an eukaryotic coexpression plasmid (pIRES-CEA/IL-2)containing two expression unites of CEA and IL-2 gene connected with internal ribosome site was constructed.ResultsThe coexpression plasmids were transformed into COS7 cells and expression of two proteins were demonstrated by ELISA, and flow cytometer and elecsy.CEA and IL-2 were (23.73?0.26)ng/ml,and(20.17?0.13)ng/ml respectively.ConclusionsThe eukaryotic expression plasmids pIRES-CEA/IL-2 could be successfully constructed and transformed into COS7 cells.Expression of two proteins were demonstrated with no difference on expression.

Objective To compare the predictive value of 5 scoring systems for hemorrhagic transformation risk after intravenous thrombolysis in patients with acute ischemic stroke (AIS) in different therapeutic windows.Methods A single-center and retrospective study was performed for 243 AIS patients who underwent intravenous thrombolysis using recombinant tissue plasminogen activator (rt-PA) in different therapeutic windows in our department during January 2014 and December 2016.Five scoring systems,including HAT model (hemorrhage after thrombolysis),MSS model (multicenter stoker survey),GRASPS model (glucose at presentation,race,age,sex,systolic blood pressure at presentation,severity of stroke at presentation),SEDAN model (baseline blood sugar,early infarct signs,hyperdense cerebral artery sign on admission CT,age,NIHSS on admission),and SITS model (safe implementation of thrombolysis in strokemonitoring study) were used to evaluate the risks for hemorrhagic transformation.The relationships between the 5 scoring systems and incidence rate of hemorrhagic transformation were analyzed among the patients in different therapeutic windows.The predictive values of the 5 scoring systems were compared using the areas (AUC) under the receiver operating characteristic (ROC) curve.Results When the AIS patients were treated by intravenous thrombolysis within 3 h,the AUC of GRASPS and HAT models were 0.698 and 0.619,respectively,higher than those of the other 3 systems.When the therapeutic window was between 3 to 4.5 h,HAT model and SEDAN model had highest AUC (0.719,0.744) than the other 3 systems (P ＜0.05).When the windows were ＞4.5 ～6 h,the HAT model had the highest AUC (0.676).Conclusion The 5 scoring systems show better predictive value for hemorrhagic transformation after intravenous thrombolysis.For the therapeutic window within 4.5 h,HAT model presents best predictive value than the other 4 scoring systems.