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Classical swine fever (CSF), one of OIE-listed diseases, is a highly contagious and economically important disease of pigs. Classical swine fever virus (CSFV) is the causative agent of CSF. The capsid (C) protein and the glycoproteins Erns, E1 and E2, are structural components of the virus. E2 is the most immunogenic protein of the CSFV glycoproteins, inducing neutralizing antibodies that provide protection against lethal CSFV challenge. In a previous study, we developed a murine MAb HQ06 against the E2 protein of CSFV. In this study, the variable region genes from HQ06 and constant regions gene of swine antibody are fused and cloned into the eukaryotic expression vectors to establish a cell line which can stably express a chimeric porcinized MAb (cHQ06) against E2 in CHO cell. The purified cHQ06 antibody protein was determined to be successfully generated, which exhibited high reactivity between cHQ06 and the E2 protein of CSFV by enzyme-linked immunosorbent assay (ELISA) and Western blotting. More importantly, we investigated the neutralizing activity of cHQ06 against CSFV. In conclusion, this study generated cHQ06 for efficient and stable production which can be used against to develop novel diagnostic assays, investigate the structure and function of the E2 protein and generate novel preparations of diagnosis and treatment.
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Objective:To construct and express anti CD3/anti CD20 Diabody and identify its biological activity.Methods:PCR and overlap PCR were used to construct anti CD3/anti CD20 Diabody.DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide.The product was purified by affinity chromatography and analyzed by both the detection of Western blot and size exclusion chromatography;its antigen binding activity was examined by FACS and rosetting assay.Results:The data of DNA sequence showed that the anti CD3/anti CD20 Diabody was corrected.The Diabody was recovered in high yield(up to 1 mg/ml) after E taq purification and predominantly(90%) as a dimer.The Diabody binded Jurkat cells(CD3 +) and Daudi cells (CD20 +),respectively.Furthermore,the Diabody was capable of simultaneous binding to Jurkat cells and Daudi cells as shown by cellular rosetting.Conclusion:The anti CD3/anti CD20 BsF(ab') 2 was first recast into the Diabody format and succeeded to obtain high level expression.The results of some biological activity experiments indicated that the Diabody could bind to Jurkat cells and Daudi cells.
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Objective:To highly express the secretory gene engineering anti CD20F(ab') 2 in the E.coli,simplify working processes and enhance the bioactivity of the antibody fragment.Methods:Using the single factor analysis to optimize the ferment parameters.A culture method which most suit for E.coli secreting anti CD20F(ab') 2 was selected.The anti CD20F(ab') 2 was extracted from periplasm then purified it using the strptococal protein G affinity colume and the S200 HR size exclusion chromatography.The activity of anti CD20F(ab') 2 inhibiting the growth of cell Daudi in vitro was eamined using MTT.Results:Using the optimized culture method,the yield of anti CD20F(ab') 2 was distinctly enhanced from 1.9～2.2 mg/L to 3.7～4.3 mg/L and the proportion of anti CD20F(ab') 2 in all the extract also was improved from 9.7～13.2% to 38.1%～46.8%.After the S200 HR size exclusion chromatography,the purity of anti CD20F(ab') 2 could exceed 85%.The outcome of MTT exmiination showed the IC 50 of anti CD20F(ab') 2 and Fab' were 14.6 ?g/ml and 39.5 ?g/ml,respectively.Conclusion:The gene engineering anti CD20F(ab') 2 could highly express in the E.coli by using the optimized culture method.The anti CD20F(ab') 2 inhibited the growth of Daudi cells in vitro more strong than anti CD20Fab'.
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Background and purpose:Bispecific antibody is a special pattern of genetic engineering antibody.It possesses 2 antigen binding sites, one directed at the effecter cells and the other at the target cells which are always the tumor cells.This enables the effecter cells to recruit to the targets efficiently and exert cytotoxic effect.The experiment was aimed to construct the expression vector of anti-CD3?anti-CD19 bispecific antibody, purification and analyzing the activity of the diabody.Methods:Applyed PCR and overlap PCR to construct the expression vector of anti-CD3?anti-CD19 bispecific antibodies, transformed it into E.coli 16C9 for expression.Purified the diabody by His-tag affinity chromatography and confirmed by 12% SDS-PAGE and Western blot.Antigen binding activity of the diabody was analyzed by FACS.Results:The DNA sequence of the expression vector containing anti-CD3?anti-CD19 fragment was comfirmed.The yield of purified diabody was up to 5 mg/L.The molecular weight of diabody was correctly indicated by SDS-PAGE and Western blot.The diabody could specifically bind to CD19+ Raji cells and CD3+ Jurkat cells while competitively inhibit binding of HIT3a and HIT19a to the cells.Conclusion:Successfully constructed the expression vector of anti-CD3?anti-CD19 bispecific antibodies, the diabody acquired by the method could be produced with high level expression and also had high specific affinity with CD19+ Raji cells and CD3+ Jurkat cells.
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Aim To construct human phage single-chain antibody library associated with esophageal cancer and to screen the specific scFv against Eca109 cells from the liberary. Methods Metastatic periesophageal lymph nodes of esophageal cancer patients were used as the B cells source, the total RNA of these B cells was extracted and prepared as the template of RT-PCR. First, we screened graticulely two pairs of primers of the heavy and light regions separately, then the V_H and V_L fragments were first amplified from the cDNA by the polymerase chain reaction (PCR). Second, the V_H-linker and V_L-linker were amplified from the V_H and V_L fragments. Last, the V_H-linker and V_L-linker were assembled into scFv gene fragments by SOE-PCR,and then Sfi I and Not I restriction site were inlet in it. ScFv gene was cloned into the pCANTAB-5E phagemid. Phagemids were introduced into E.coli TG1 by electrotransformation, followed by rescue of antibody-expressing phage using M13K07 helper-phage superinfection. Recombinant scFv phage library was constracted and PCR was used to identify the insert ratio of scFv antibodies library. Results of SfiI/Not I double digestion reaction positive insert clone were identified by 1.5% agarose gel electrophoresis. The phage library was panned with NHEEC and Eca109 cancer cells in suspension for four rounds. Strongly positive recombinant phage clones were used to infect E.coli HB2151. Expression of soluble scFv was induced by IPTG. Soluble scFv from periplasm were purified by affinity chromatography and identified by SDS-PAGE and Western blot. Cell ELISA , immunohistochemical staining and immunocytochemical staining were used to identify the activity of the soluble scFv. Results The result of agarose gel electrophoresis showed that total RNA of these B cells had two bands of 28 S and 18 S. The size of V_H fragment is about 450 bp,V_L fragment is about 350 bp and scFv is about 850 bp. The competence is 108 cfu??g-1 pUC18 DNA. Randomly digestive reac-tion showed that the positive insert ratio was 91.7% (22/24). After four rounds of panning, the fourth phage yield is 141 times as much as that of the first one. SDS-PAGE and Western blot showed that the MW of the soluble scFv was about 30 ku and the brand of 30 ku was stained. Immunohistochemical staining showed strong stainning of the tissue of esophageal cancer, but not the liver and gastric cancer tissue. Immunocytochemical staining showed significant staining of the esophageal cancer line Eca109. The result of cell ELISA assay revealed that soluble scFv had highly specific and could combined with Eca109 cells, but not with BGC-823 and NHEEC. Conclusion A human scFv phage display library associated with esophageal cancer has been constructed successfully and the specific scFv antibody against Eca109 has been identified from the liberary.
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Objective To investigate the effect of an anti-HER-2 engineered antibody,chA21,on the expression of MMP-2 and TIMP-2 in human ovarian cancer SKOV3 cells that highly express HER-2.Methods After exposure to chA21 at concentrations of 6 mg/L and 12 mg/L for 36 hours,respectively,the expression of MMP-2 and TIMP-2 proteins was detected by immunocyctochemistry.SKOV3 cells were inoculated into nude mice to establish animal models.The mice were respectively administered with normal saline and chA21(30 mg/kg) via injection twice a week for 6 consecutive weeks,and then were killed after 44 days' administration of the drugs.Immnohistochemical staining of MMP-2 and TIMP-2 was observed on tissue microarray sections.Results After exposed to chA21,TIMP-2 protein was increased(P
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Objective To study the anti-tumor effect of anti-HER-2 engineering antibodies chA21 and Herceptin on nude mice xenografts of human ovarian cancer SKOV3 cells and explore its mechanism.Methods An animal model with human ovarian cancer SKOV3 cells involved in nude mice was established and the mice were randomized into 3 groups: normal saline(NS),chA21 and Herceptin.The mice were respectively administrated with Herceptin(30mg/kg) and chA21(30mg/kg) via caudal vein injection twice a week for consecutive 6 weeks,and then were killed after 44 days adminstration of the drugs.The volumes of the xenografts were measured twice a week.The tumor weight and inhibition ratio were measured after mice were killed.Ki67 and NF?B expression in the three groups was quantificationally analyzed by immunohistochemistry on tissue microarray sections combined with a micro-image analysing system.Results The growth of xenografts of human ovarian cancer SKOV3 cells in nude mice was significantly inhibited by either Herceptin or chA21. Both Ki-67 labeling indices and NF?B levels in chA21 and Herceptin groups were lower than those in the control(P