Intracranial delivery of human Fc-deleted antibody specific to amyloid-beta peptide (A beta, anti-A beta single-chain Fv, scFv) via adeno-associated virus (AAV) inhibits amyloid deposition in transgenic mice. However, the effects of AAV-mediated Fc-deleted antibody on animal behavior remain unclear. In this study, the anti-A beta scFv antibody gene, isolated from phage display, was fused to the 5' end of the scFv antibody gene for antibody secretion by 2 rounds of polymerase chain reaction amplification. The fused antibody cDNA was cloned into a pSNAV2 plasmid under the control of the cytomegalovirus promoter. The sequence verified expression vector pSNAV2/scFv was transferred to BHK-21 cells, and stable transfected BHK-21/scFv cells were established by G418 selection and infected with the recombinant herpes simplex virus rHSV/repcap for AAV production. Recombinant AAV was injected into the left quadriceps femoris of PDAPP transgenic mice. After 3 months, Morris water-maze results confirmed significantly improved cognitive function in a mouse model of Alzheimer's disease.

OBJECTIVE To investigate the therapeutic effect of recombinant adeno-associated virus carrying anti-amyloid β peptide single-chain antibody gene on Alzheimer's disease (AD) in animal models. METHODS The recombinant adeno-associated viruses were injected to the leg muscle of mutant amyloid precursor protein transgenic AD mice. The latency of the mice in Morris water maze was tested before and 3, 7, 10 months after drug administration. The animal brains were harvested 10 months after drug administration and sectioned for amyloid plaques staining. RESULTS The learning and memory abilities of AD model mice were improved significantly 3 months after gene drug administration. Ten months after gene therapy, the numbers of amyloid plaque in hippocampus of model mice decreased. CONCLUSION The adeno-associated virus carrying anti-amyloid β peptide single-chain antibody gene has therapeutic effect on AD in model mice.

Objective To derermine the affinity of site-specific labeled annexin V with 99mTc to phosphatidylserine(PS) exposed erythrocytes.Methods The annexin V fused with a metal chelating binding site was obtained from Pichia Pastoris culture and methanol induction expression.The annexin V was purified from the culture supernatant crude product by ultrafiltration.The annexin V was conjugated with 99mTc site-specifically through sodium glucoheptonic acid and SnCl2.The radioactive annexin V was added to determine its affinity to PS exposed erythrocytes.Results The calcium concentration at which half of the protein is bound to cells(EC50) is changed with varying ratio of protein to cells.The affinity was determined at a low protein/cell ratio as 33.4.Conclusion The annexin V recombinantly expressed in Pichia Pastoris shows high affinity to PS exposed erythrocytes.

阿尔茨海默病(Alzheimer's disease, AD)的发生、发展和β淀粉样肽(amyloid β peptide, Aβ)在大脑组织的过载和神经毒性密切相关~([1]).动物实验和临床测试结果均表明Aβ疫苗免疫接种或Aβ抗体的外周注射有助于清除大脑组织内过载的Aβ~([2-3]).

Objective To prepare human annexin V dimmer and make chance for its pharmacological research.Methods Pichia pastoris transformed by human annexin V gene was used in this investigation.The engineered yeast was cultured in BMGY media and induced with methanol media BMMY for secreting expression of human annexin V.The final BMMY media supernatant was adopted for preparation of human annexin V dimmer by ammonium sulfate precipitation,molecular sieve separation and ultra-filtration concentration.The purification process was monitored by SDS-PAGE and coomassie brilliant blue staining.Results Human annexin V was obtained after two days induction of pichia pastoris in BMMY media.The human annexin V dimmer was successfully prepared from culture supernant by serial ammonium sulfate precipitation,molecular sieve separation and ultra-filtration concentration.Conclusion We prepare the human annexin V dimmer(diannexin V)successfully.

Objective To produce and purify recombinant adeno-associated virus(AAV) loaded with anti-amyloid β peptide single-chain antibody gene for gene therapy of Alzheimer's disease.Methods The plasmid pSNAV2.0-Abeta-scFv was used to transform BHK-21 cell by Lipofectamine 2000 for stable package cell line establishment.The helper virus HSV1-rc/ΔUL2 was used to transfect package cell for anti-amyloid β peptide single-chain antibody gene loaded recombinant adeno-associated virus production.The chloroform-PEG/NaCl-chloroform extraction and ion-exchange chromatography were employed for recombinant AAV purification.SDS-PAGE and PCR amplification were adopted for purified virus identification.The final virus physical titer was determined by digoxin-labeled DNA probes.The effect of gene therapy was tested with transgenic mice by water maze test.Results The purity of recombinant adeno-associated virus with our target gene reach up to 98% after stable cell package and serial purification.The physical titer of the final virus was 1×1012vg/mL.The latency of treated mice in water maze test were reduced significantly.Conclusion The adeno-associated virus carrying anti-amyloidβ peptide single-chain antibody gene was produced by HSV1system and purified.The animal behavior test demonstrated the recombinant AAV waseffective in the gene therapy of Alzheimer's disease.

BACKGROUND : Studies have shown that monoclonal or polyclonol antibody injections of amyloid beta peptide are effective in removing amyloid beta peptide overload in the brain.OBJECTIVE: Based Oil Successful screening of a human single-chain fragment variable antibody specific to amyloid beta peptide. this paper aimed to express recombinant human single-chain variable antibody against amyloid beta peptide.DESIGN,TIME AND SETTING: A single sample experiment was performed at the Department of Nuclear Medicine. Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Hospital (Beijing, China) front January to July 2006.MATERIALS: Human single-chain fragment variable antibody gene against amyloid beta peptide was screened from a human phage-display antibody library.METHODS: Human single-chain fragment variable antibody gene was mutated to eliminate a BamHI restriction site and cloned into a T easy plasmid for pT-scFvA beta construction, which was identified by PCR amplification and endonuclease digestion. Plasmid pT-scFvA beta was cut by EcoR1 and NotI endonucleases, and the antibody gene was cloned into pPIC9 K plasmid to construct pPIC9K-scFvA beta expression vector, which was confirmed by gene sequencing. Linearized pPIC9K-scFvA beta was used to transform a Pichia pastoris GS115 cell line, and the recombinant was induced by 0.5% methanol to express human single-chain fragment variable antibody specific to amyloid beta peptide.MAIN OUTCOME MEASURES: Protein electrophoresis Was used to identify PCR products. gene sequencing was used to verify the pPIC9K-scFvA (R) sequence, and SDS-PAGE Was used to detect recombinant expression of human single-chain fragment variable antibody specific to amyloid beta peptide in Pichia pastoris.RESULTS: Gene sequencing confirmed pPIC9K-scFvA beta orientation. Recombinants were obtained by linearized pPIC9K-scFvA beta transformation. After induction with 0.5% methanol. the recombinant yeast cells secreted proteins of 33-ku size.CONCLUSION: The expression vector pPIC9K-scFvA beta was successfully constructed. Human single-chain fragment variable antibody specific to amyloid beta peptide was recombinantly expressed in Pichia pastoris.

Objective To prepare a novel annexin V and conjugate it with fluorescein isothiocyanate and test their binding ability to phosphatidylserine(PS)exposed erythrocytes.Methods The annexin V fused with an metal chelating binding site was obtained from pichia pastoris culturing and methanol induction expression.The annexin V was purified from the culture supernatant crude product by ion-exchange chromatography and ammonium sulfate precipitation.The annexin V was conjugated with fluorescein isothiocyanate in boric buffer and the conjugate annexin V-FITC was purified by ion-exchange chromatography.Sheep red blood cells were treated with Glutaraldehyde to expose membrane PS.The annexin V-FITC binding to PS exposed erythrocytes was investigated by fluorescent microscopy.Results The novel annexin V was purified and concentrated from pichia pastoris culture by ion-exchange chromatography and ammonium sulfate precipitation.After conjugating with FITC,the annexin V was found to bind PS exposed erythrocytes by fluorescent microscopy.Conclusion The annexin V expressed recombinantly in pichia pastoris retains the binding ability to PS exposed erythrocytes and is applicable to apoptosis detection in vitro.

OBJECTIVE:To prepare 99Tcm-annexinV and evaluate its application in the early prediction of therapeutic effect of chemical agent on lung cancer.METHODS:Annexin V was obtained by recombinant pichia pastoris expression, ammonium sulfate precipitation, and size-exclusion chromatography. The purified protein was labeled with 99Tc(m) at the N-terminal site by stannous chloride reduction and purified by desalting. The labeling yield and radiochemical purity of 99Tc(m)-annexinV were determined by instant thin-layer chromatography. The biological activity of 99Tc(m)-annexinV was tested by phosphatidylserine-exposed erythrocytes bound radioactivity counting. The lung cancer mice models were established by inoculating LA795 cells to right flank of 615 mice subcutaneously and tumor tissue transplantation. The biodistribution of 99Tc(m)-annexinV in lung cancer mice models were investigated at 6, 12, 24, and 48 h after cyclophosphamide administration.RESULTS:The annexin V was secreted from pichia pastoris and purified by ammonium sulfate precipitation and size-exclusion chromatography with high yield. The annexin V could be labeled at room temperature with 50.2% radioactivity yield. The radiochemical purity of 99Tc(m)-annexinV reached up to 93.9% with intact biological activity. The biodistribution analysis demonstrated that 99Tc(m)-annexinV was excreted from kidney. The uptake of 99Tc(m)-annexinV at tumor reached maximum 48 h after cyclophosphamide administration while tumor to muscle ratio was 6.34 and tumor to blood ratio was 4.09.CONCLUSIONS:99Tc(m)-annexinV derived from pichia pastoris was successfully prepared. It is useful in predicting the therapeutic effect of chemical agent on lung cancer.

Objective: To explore the biological activity of recombinant human single-chain antibody against amyloid β peptide in vitro. Methods: Human single-chain antibody against amyloid β peptide was obtained from recombinant bacteria. The antigen-binding activity of this antibody was measured by enzyme-linked immunosorbent assay (ELISA) and competitive ELISA. Human neuroblastoma SH-SY5Y cells were used as cell models to test the protective role of human single-chain antibody against amyloid β peptide. Results: Recombinant human single-chain antibody was mainly located in the insoluble inclusion bodies of bacteria. The antibody was dissolved by urea and purified by metal affinity chromatography as active form to bind synthetic amyloid β peptide 40 or amyloid β peptide 42. The improvement of the survival rates of human neuroblastoma cells was significantly superior in amyloid peptide 42 plus equimolar antibody group than in amyloid peptide 42 group (P < 0.05), and was significantly superior in the amyloid peptide 40 plus equimolar antibody group than in amyloid peptide 40 group (P < 0.01). Conclusion: The recombinant human single-chain antibody against β amyloid peptide 40 from E. coli can partially inhibit the neurotoxicity effect of amyloid β peptide in vitro.

Objective To express anti-amyloid β peptide 40 human monoclonal antibody in baculovirus insect system, so as to provide a candidate antibody therapeutics for Alzheimer’s disease patients. Methods pAC-VH-VL expression palsmid was constructed by cloning heavy and light chain variable region separately into pAC-L-CH3 MCS. The expression palsmids were co-infected with BaculoGold virus into SF9 cells and the IgG expression was detected by immunoflurescence method. The culture supernatant of baculovirus-infected cells was loaded on pre-equilibrated protein A Sepharose and the eluted antibody was subjected to SDS-PAGE analysis.Results The expression plasmid pAC-VH-VL was constructed as expected. The infected insect cells secreted human antibody after co-infected with expression plasmid and helper virus. Anti-amyloid β peptide 40 human monoclonal antibody was purified from culture supernatant of infected insect cells by protein A affinity chromatography. Conclusions The constant region of human antibody was fused to carboxyl end of anti-Aβ40 human antibody variable region by gene engineering tools. The whole anti-Aβ40 human monoclonal antibody was expressed in baculovirus insect cells system and purified by protein A affinity chromatography.

OBJECTIVETo generate a sensitive tool for noninvasive monitoring of a therapeutic gene vasostatin.METHODSWe fused the bioluminescent reporter gene firefly luciferase to the therapeutic transgene vasostatin and ensured that these two proteins would not interrupt each other and kept their own natural character.RESULTSWe therefore examined clones of PC3 cells stably expressing fusion gene and positive controlfluc with bioluminescence. In vivo imaging of PC3-Fluc subcutaneous tumors showed that the mean tumor bioluminescence increased in animals over several weeks.CONCLUSIONNoninvasive monitoring facilitates the detection of gene expression in vivo and in vitro.

Selenoprotein K (SelK) is a newly identified selenoprotein. We showed that selenium incorporation into SelK was dependent on the 3′UTR of SelK mRNA. Sec insertion sequence (SECIS) RNA binding assays demonstrated that human SBP2 bound to the SelK SECIS element through the conserved non‐Watson–Crick base pair quartet but not the AAT motif. Examination of the expression pattern revealed that human SelK mRNA was highly expressed in heart. Immunofluorescence analysis showed that SelK localized to the endoplasmic reticulum. Using SelK recombinant adenovirus, we found that overexpression of SelK attenuated the intracellular reactive oxygen species level and protected cells from oxidative stress‐induced toxicity in cardiomyocytes. Our findings indicated that SelK is a novel antioxidant in cardiomyocytes and is related to the regulation of cellular redox balance.

Objective To express human single-chain antibody against amyloidβpeptide 40 in pichia pastoris for passive immuniza- tion to Alzheimer's disease patients.Methods Human single-chain antibody gene from a phage display library was mutated to obliterate BamHI site and cloned into pAO815 plasmid for pT-scFvAβ40 construction which was identified by PCR amplification and endonuclease digestion.Vector pT-scFvAβ40 was cut by EcoRI and NotI endonucleases and the antibody gene was cloned into pPICgK plasmid to con- struct pPIC9K-scFvAβ40 expression vector which was confirmed by gene sequencing.Linearized pPIC9K-scFvAβ40 was used to trans- form pichia pastoris GS115 and the recombinant was induced by 0.5% methanol to express human single - chain antibody against amyloidβpeptide 40.Results Gene sequencing confirmed pPIC9K-scFvAβ40 orientation.Recombinants were obtained by linearized pPICgK - scFvAβ40 transformation.After inducing with 0.5% methanol,the recombinant secreted proteins with 33 kD size.Conclusions Expression vector pPIC9K-scFvAβ40 was constructed successfully.Human single-chain antibody against amyloidβpeptide 40 was recombinantly expressed in pichia pastoris.

我们知道,对疾病要早发现、早诊断、早治疗.而病人往往是在身上出现症状以后才会就医.但此时病人机体早就在分子水平上发生了一系列生化改变,这是人不能感知的,也不能通过肉眼观察到.到表现出症状时,已经错过了最佳治疗时机,不仅医疗费用大大增加,而且治愈的机会也少了许多.

OBJECTIVE The synthesis, biodistribution, and animal imaging of 99mTc- hydrazinonicotinamide-folate (99mTc-HYNIC-Folate) were studied as a folate receptor-targeted tumor imaging agent. METHODS HYNIC-Folate was synthesized by a muti-step reaction and radiolabeled with 99mTc using tricine and trisodium phenylphosphine-3, 3', 3"-trisulfonate (TPPTS) as coligands. The radiochemical purity and stability of 99mTc HYNIC-Folate was measured. The biodistributions of 99mTc-HYNIC-Folate in normal mice and tumor-bearing mice were detected. Whole-body gamma imaging was performed using an athymic mouse tumor xenograft model. RESULTS The ligand HYNIC-Folate was successfully synthesized and characterized by hydrogen nuclear magnetic resonance (1HNMR) and mass spectrometry (MS). The radiochemical purity of 99mTc-HYNIC-Folate was 96% under optimal conditions. Data from gamma scintigraphy and the biodistribution in tumor-bearing mice showed that 99mTc-HYNIC-Folate predominantly accumulated in tumor, its uptake rate per gram tissue alpham was 5. 620+/- 0. 753. The uptakes of 99mTc-HYNIC-Folate in the other non-target tissues were very low, except it was high in the kidneys ( am was 41. 959 +/-6. 759) . CONCLUSION 99mTc-HYNIC-Folate has the potential to be used as a noninvasive radiodiagnostic imaging agent for the detection of folate receptor-positive human cancers.

AIM: To express and purify human single-chain antibody against amyloid β peptide 40 from bacteria system for passive immunization to Alzheimer disease patients, which represented a promising therapeutic approach. METHODS: The experiment was carried out in the Laboratory of Nuclear Medicine between August 2004 and March 2005. Human single-chain antibody gene from a phage display library was cloned into pET22b (+) plasmid expression vector and transformed competitive BL21-gold cells. After inducing the transformed cells with 0.5 mmol/L isopropyl-β-D-thiogalactoside (IPTG). The insoluble pellet was dissolved in buffer and loaded to pre-equilibrated Ni2+-NTA-resin. The binding protein was re-natured and eluted from the resin with imidazole. The protein eluted was identified by Western blotting and enzyme-linked immunoadsordent assay (ELISA) with mouse monoclonal antibody and HRP-labeled goat anti-mouse antibody. Concentration of the human single-chain antibody was determined by Branford method.RESULTS: ① Anti-amyloid β 40 human single-chain antibody gene consisted of about 750 bp and the relative molecular mass of expressed protein was about 33 000. ② The recombinant human single-chain antibody from bacteria inclusion body was purified by affinity purification as single band. ③ ELISA demonstrated that the antigen-binding activity of the purified antibody was higher than the control significantly (A595 nm: 0.59±0.06, 0.12±0.02, P < 0.05), it was indicated that the human single-chain antibody purified from bacteria inclusion body maintained the immune activity of binding antigen. The concentration of purified amyloid β 40 single-chain antibody was 96 mg/LCONCLUSION: Anti-amyloid β 40 human single-chain antibody gene can be expressed in Escherichia coli system and the recombinant antibody is purified with high purity by affinity chromatography.

移植肿瘤模型的研究表明,化疗和放疗对肿瘤的杀伤效应是通过对细胞凋亡过程的迅速诱导来实现的[1].