Protein expression and preparation of polydonal antibody of AD-004 and study on its expression in the adrenal and testis

Objective To prepare rabbit antibody against mouse AD-004 by AD-004 expressed in the prokaryotic expression system and to identify its distribution in the testis and adrenal. Methods The full-length cDNA of mouse AD-004 was cloned into PET28 plasmid, and the protein was induced in E. coli BL21 bacteria by adding IPTG and then purified by Ni2 -NTA column. The purified protein was used as an immunogene to prepare polyclonal     

Relationship between expression of Smac and Survivin and apoptosis of primary hepatocellular carcinoma

Introduction Disturbance of the regulation of cell apoptosis can lead to cell over-proliferation, and decrease of apoptosis can result in tumorigenesis ortumor development. The apoptotic signaling pathway is regulated by a variety of factors and is based on the balance between cell death and survival factors.[1, 2] The central players in apoptosis are caspases, and one important route to activation of the caspases involves the translocation of cytochrome C (Cyt-C) from mitochondria to cytosol…     

Cloning and expression and immunogenicity of Helicobacter pylori BabA_2 gene

AIM:To construct a recombinant strain which expresses BabAof Helicobacter pylori(Hpylon)and to study the immunogenicityof BabA.METHODS:BabA_2 DNA was amplified by PCR and insertedinto the prokaryotie expression vector pET-22b( )andexpressed in the BL21(DE3)E.coli strain.Furthermore,BabA immunogenicity was studied by animal test.RESULTS:DNA sequence analysis showed the sequenceof BabA_2 DNA was the same as the one published by GenBank.The BabA recombinant protein accounted for 34.8% of thetotal bacterial protein.The serum from Hpyloriinfectedpatients and Balb/c rniced immunized with BabA itself couldrecognize rBabA.CONCLUSION:BabA recombinant protein may be an potentialvaccine for control and treatment of Hpyloriinfection.     

Construction of prokaryotic expression system of ItB-ureB fusion gene and identification of the recombinant protein immunity and adjuvanticity

AIM:To construct ItB-ureBfusion gene and its prokaryoticexpression system and identify immunity and adjuvanticityof the expressed recombinant protein.METHODS:The ureB gene from a clinical Helicobacterpylori(H pylon) strain Y06 and the ItB gene from Escherichiacoli (E.coli) strain 44851 were linked into ItB-ureB fusiongene by PCR.The fusion gene sequence was analyzedafter T-A cloning.A prokaryotic recombinant expressionvector pET32a inserted with ItB-ureB fusion gene (pET32a-ItB-ureB) was constructed.Expression of the recombinantLTB-UreB protein (rLTB-UreB) in E.coli BL21DE3 inducedby isopropylthio-β-D-galactoside (IPTG) at differentconcentrations was detected by SDS-PAGE.Western blotassays were used to examine the immunoreaction of rLTB-UreB by a commercial antibody against whole cell of H pyloriand a self-prepared rabbit anti-rUreB serum,respectively,and determine the antigenicity of the recombinant proteinon inducing specific antibody in rabbits.GM_1-ELISA wasused to demonstrate the adjuvanticity of rLTB-UreB.Immunoreaction of rLTB-UreB to the UreB antibody positivesera from 125 gastric patients was determined by using ELISA.RESULTS:In comparison with the corresponding sequencesof original genes,the nucleotide sequence homologies ofthe cloned ItB-ureB fusion gene were 100%.IPTG withdifferent dosages of 0.1-1.0 mmol/L could efficiently inducepET32a-ItB-ureB-E.coli BL21DE3 to express the rLTB-UreB.The output of the target recombinant protein expressedby pET32a-ureB-E.coli BL21DE3 was approximately 35%of the total bacterial proteins,rLTB-UreB mainly presentedin the form of inclusion body.Western blotting resultsdemonstrated that rLTB-UreB could combine with thecommercial antibody against whole cell of H pylori andanti-rUreB serum as well as induce rabbit to producespecific antibody.The strong ability of rLTB-UreB bindingbovine GM_1 indicated the existence of adjuvanticity of therecombinant protein.All the UreB antibody positive serafrom the patients (125/125) were positive for rLTB-UreB.CONCLUSION:A recombinant prokaryotic expression system with high expression efficiency of the target fusiongene ItB-ureB was successfully established.The expressedrLTB-UreB showed qualified immunogenicity,antigenicityand adjuvanticity.All the results mentioned above laid afirm foundation for further development of H pylori geneticallyengineered vaccine.     

Repression of α-synuclein expression and toxicity by microRNA-7

α-Synuclein is a key protein in Parkinson''s disease (PD) because it accumulates as fibrillar aggregates in pathologic hallmark features in affected brain regions, most notably in nigral dopaminergic neurons. Intraneuronal levels of this protein appear critical in mediating its toxicity, because multiplication of its gene locus leads to autosomal dominant PD, and transgenic animal models overexpressing human α-synuclein manifest impaired function or decreased survival of dopaminergic neurons. Here, we show that microRNA-7 (miR-7), which is expressed mainly in neurons, represses α-synuclein protein levels through the 3′-untranslated region (UTR) of α-synuclein mRNA. Importantly, miR-7-induced down-regulation of α-synuclein protects cells against oxidative stress. Further, in the MPTP-induced neurotoxin model of PD in cultured cells and in mice, miR-7 expression decreases, possibly contributing to increased α-synuclein expression. These findings provide a mechanism by which α-synuclein levels are regulated in neurons, have implications for the pathogenesis of PD, and suggest miR-7 as a therapeutic target for PD and other α-synucleinopathies.     

Immunohistochemical study of epidermal and dermal expression of Bcl-X, Bcl-2 and bax in psoriasis.

Objective: To investigate the regulation of cell proliferation and apoptosis in psoriasis. Methods: The expressions of Bcl-X, Bcl-2 and Bax were studied with immunohistochemical technique (SP) in the lesional and non-lesional psoriatic skin. Results: There were significant overexpressions of Bcl-X in all layers of epidermis, inflammatory cells and vascular endothelia in dermis;     

Construction of the pIRES2-ZsGreen1 eukaryotic expression vector of factor Ⅸ gene and expression in HEK-293 cells

正Objective To construct p IRES2-ZsG reen1/FⅨexpression vector,using the pcDNA/FⅨplasmid containing FⅨcDNA as template,and expressing in HEK-293cells.Methods The total ORF of FⅨgene was amlified from pcDNA/FⅨplasmid,then the amplified fragment was clonded into the p IRES2-ZsG reen1 vector using the     

Glucocorticoids facilitate astrocytic amyloid-β peptide deposition by increasing the expression of APP and BACE1 and decreasing the expression of amyloid-β-degrading proteases

In most cases, the molecular mechanism underlying the pathogenesis of sporadic Alzheimer's disease (AD) is unknown. Elevated basal cortisol levels in AD patients suggest that glucocorticoids (GC) may contribute to the development and/or maintenance of AD. Amyloid plaques are the hallmark of AD, and they are considered to play an early role in the AD process. However, little is known about how their formation is regulated by stress and GC. Astrocyte accumulation is one of the earliest neuropathological changes in AD. Here, we report that GC elevated amyloid-β (Aβ) production in primary cultures of astrocytes by increasing amyloid precursor protein (APP) and β-site APP-cleaving enzyme 1 gene expression. Notably, GC administered to normal, middle-aged mice promoted the expression of APP and β-site APP-cleaving enzyme 1 in astrocytes, as determined by double immunofluorescence. Additionally, confocal microscopy and ELISA revealed that GC markedly reduced Aβ degradation and clearance by astrocytes in vitro, indicating a decreased neuroprotective capacity of the astrocytes. This may have been due to the decrease of several Aβ-degrading proteases, such as insulin-degrading enzyme and matrix metalloproteinase-9. These effects occurred through the activation of GC receptors. Taken together, our results demonstrate that GC can enhance the production of Aβ, reduce its degradation in astrocytes, and provide a molecular mechanism linking stress factors to AD. Our study suggests that GC can facilitate AD pathogenesis and that reducing GC in the elderly and early AD patients would be beneficial.     

Clinicopathologic analysis of esophageal and cardiac cancers and survey of molecular expression on tissue arrays in Chaoshan littoral of China

AIM:To investigate clinical and pathologic data ofesophageal carcinoma(EC)and cardiac carcinoma(CC)among residents in Chaoshan region of China.METHODS:Clinical and pathologic data of 9 650 patientswith EC and 4 173 patients with CC in the Chaoshanpopulation were collected and analyzed.Moreover,Chaoshan esophageal carcinoma tissue arrays were madefor high-throughput study.RESULTS:Male to female ratio was 3:1 in patients withEC and 4.75:1 in CC.The average age of the occurrenceof EC was 54.6 years,and of CC was 58.1 years.For bothEC and CO,age at diagnosis was a little younger inChaoshan region than in most other areas.The mostcommonly affected site of esophageal carcinoma was themiddle third of esophagus(72.0%);the second was thelower third(15.3%).The main gross type of esophagealcarcinoma was ulcerative type(dl.50%);the medullary typewas the second(39.6%).Squamous cell carcinoma accountedfor the overwhelming majority of esophageal cancer(96.4%);adenocarcinoma accounted for the overwhelming majorityof cardiac carcinoma(94.5%).Chaoshan esophagealcarcinoma tissue arrays were easily for high-throughputstudy,and tissue cores with a diameter of 1.5 mm couldbetter keep more structure for molecular expression study.CONCLUSION:Both EC and CC are common in males.The average occurrence age of EC and CC is younger inChaoshan than in most other regions of China.The mostcommonly affected site of esophageal carcinoma was themiddle third of esophagus(72.0%).Squamous cell carcinoma accounted for the overwhelming majority ofesophageal cancer;adenocarcinoma accounted for theoverwhelming majority of cardiac carcinoma.Tissue arraystechnology is applicable for rapid molecular profiling oflarge numbers of cancers in a single experiment.