AIM:To assess BGC823 gastric cancer(GC) cell metastasis after knockdown of liver-intestine cadherin(CDH17) and the therapeutic value of CDH17-RNAilentivirus in vivo.METHODS:We evaluated primary tumor growth and assess...AIM:To assess BGC823 gastric cancer(GC) cell metastasis after knockdown of liver-intestine cadherin(CDH17) and the therapeutic value of CDH17-RNAilentivirus in vivo.METHODS:We evaluated primary tumor growth and assessed local infiltration and systemic tumor dissemination using an orthotopic implantation technique.The therapeutic value of CDH17 knockdown was examined by intratumoral administration of CDH17-RNA interference(RNAi)-lentivirus in an established GC tumor xenograft mouse model.Furthermore,a comparative proteomic approach was utilized to identify differentially expressed proteins in BGC823 and lenti-CDH17-miRneg cells following CDH17 knockdown.RESULTS:Metastases in the liver and lung appeared earlier and more frequently in animals with tumors derived from BGC823 or lenti-CDH17-miR-neg cells than in tumors derived from lenti-CDH17-miR-B cells.Average tumor weight and volume in the CDH17-RNAi-lentivirus-treated group were significantly lower than those in the control group(tumor volume:0.89 ± 0.04 cm 3 vs 1.16 ± 0.06 cm 3,P < 0.05;tumor weight:1.15 ± 0.58 g vs 2.09 ± 0.08 g,P < 0.05).Fifteen differentially expressed proteins were identified after CDH17 silencing in BGC823 cells,including a variety of cytoskeletal and chaperone proteins as well as proteins involved in metabolism,immunity/defense,cell proliferation and differentiation,cell cycle,and signal transduction.CONCLUSION:Our data establish a foundation for future studies of the comprehensive protein expression patterns and effects of CDH17 in GC.展开更多
Adsorption reactions between surfaces of nanodiamond and nanosilica with diameter of 100 nm prepared as suspension solutions of 0.25μg/μL and lysozyme molecule with different concentrations of 7 mmol/L PPBS at pH=7,...Adsorption reactions between surfaces of nanodiamond and nanosilica with diameter of 100 nm prepared as suspension solutions of 0.25μg/μL and lysozyme molecule with different concentrations of 7 mmol/L PPBS at pH=7, 9, 11, and 13 have been investigated by fluores- cence spectroscopy. Adsorption reaction constants and coverages of lysozyme with different concentrations of 0-1000 nmol/L under the influences of different pH values have been ob- tained. Helicities and conformations of the adsorbed lysozyme molecules, free spaces of every adsorbed lysozyme molecule on the surfaces of nanopartieles at different concentrations and pH values have been deduced and discussed. The highest adsorption capabilities for both sys- tems and conformational efficiency of the adsorbed lysozyme molecule at pH=13 have been obtained. Lysozyme molecules can be prepared, adsorbed and carried with optimal activity and helicity, with 2 and 10 mg/m2 on unit nanosurface, 130 and 150 mg/g with respect to the weight of nanoparticle, within the linear regions of the coverages at around 150-250 nmol/L and four pH values for nanodiamond and nanosilica, respectively. They can be prepared in the tightest packed form, with 20 and 55 mg/m2, 810-1680 and 580-1100 mg/g at threshold concentrations and four pH values for nanodiamond and nanosilica, respectively.展开更多
Snake toxin Calciseptine as a natural antagonist of L-type calcium channel has potential drug values, but its structural information remains unknown. Here, we report the total chemical synthesis of Calciseptine by usi...Snake toxin Calciseptine as a natural antagonist of L-type calcium channel has potential drug values, but its structural information remains unknown. Here, we report the total chemical synthesis of Calciseptine by using hydrazide based native chemical ligation. The crystal structure of Calciseptine was determined by racemic protein crystallography technique. Compared to the structure of its homologous family protein, we found that Calciseptine is adopting a typical three-finger structure.展开更多
Epilepsy is one of the most common neurological disorders characterized by epileptic seizures.The anti-epileptic drugs(AEDs)are the main form of treatment for people with epilepsy.Classically,people thought that AEDs ...Epilepsy is one of the most common neurological disorders characterized by epileptic seizures.The anti-epileptic drugs(AEDs)are the main form of treatment for people with epilepsy.Classically,people thought that AEDs modify the activities of ion channels to suppress epileptic seizures.However,accumulating evidence suggests that targeting at ion channels cannot completely account for the numerous effects of the AEDs on its broad clinical activity in epileptic patients.In our study,proteomic methods were used to quantify the proteome of hippocampal tissues from patients who were treated or not treated by AEDs(Carbamazepine).Further bioinformatics methods,including Gene Ontology(GO)annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis,were utilized to analyze the differences between two patient groups.We found that more than 400 proteins,including metabolism and immune related proteins,had a higher expression level in the carbamazepine-treated group compared with the controls.These altered proteins were considered to be involved in many different biological pathways.Among these pathways,immune-system related pathway modulated by complement C3 and microglia was highly remarkable,which regulated the synapses elimination in physiological condition.In epilepsy,the carbamazepine induced up-regulation of complement C3 might decrease the abnormal synaptic connections between neurons and thus contribute to the therapeutic role of carbamazepine.The results of our study suggested that apart from ion channels,carbamazepine exerted numerous effects on human epileptic foci,which might be the fundamental mechanisms of AEDs for treatment,adverse-effects and pharmacoresistance of epilepsy.展开更多
Objective：To observe the effect of acupuncture on the expression of mitochondrial proteome in hippocampus of senescence-accelerated mouse prone g （SAMPg） mice models with Alzheimer disease （AD）,and to explore the...Objective：To observe the effect of acupuncture on the expression of mitochondrial proteome in hippocampus of senescence-accelerated mouse prone g （SAMPg） mice models with Alzheimer disease （AD）,and to explore the possible protective mechanism of acupuncture on mitochondria.Methods：Sixty 6-month-old male SAMP8 mice were randomly divided into an acupuncture at acupoint group,an acupuncture at non-acupoint group and a model group,20 mice in each group.The 20 male senescence-accelerated mouse/resistance 1 （SAMR1） mice of the same age were used as a normal control group.Shenshu （BL 23）,Baihui （GV 20）,Xuehai （SP 10） and Geshu （BL 17） were selected for acupuncture intervention in acupuncture at acupoint group.After an 8-week intervention,mitochondrial tissues were extracted from the hippocampus.Differentially expressed proteins were identified by subcellular organelle proteomics.Western blot was used to verify the expressions of some related proteins in hippocampal mitochondria.Results：Compared with the model group,there were 13 differentially expressed protein spots in the acupuncture at acupoint group,of which,9 were up-regulated,including neurofilament light polypeptide （NFL）,actin （cytoplasmic 1,database ID：ACTB）,tubulin beta-2A chain （TBB2A）,tropomodulin-2 （TMOD2）,pyruvate dehydrogenase E1 component subunit beta （PDHE1-β）,NADH-ubiquinone oxidoreductase 75 kDa subunit （database ID：NDUS1）,heat shock cognate 71 kDa protein （HSC71）,pyruvate dehydrogenase E1 component subunit alpha （PDHE1-α） and ATP synthase beta subunit （ATP-β）;4 were down-regulated,including glial fibrillary acidic protein （GFAP）,pyruvate dehydrogenase phosphatase 1 （PDP1）,mitochondrial-processing peptidase subunit alpha （MMP-α） and adenosine kinase （ADK）.According to the information provided in the protein database,most of the differentially expressed proteins involve the regulation of mitochondrial function and structure.The expression levels of NFL and TBB2A in the normal control group and the acupuncture at acupoint group were significantly higher than those in the acupuncture at non-acupoint group （P〈0.05）.ATP-β and NDUS1 expression levels were significantly higher in the acupuncture at acupoint group than those in the acupuncture at non-acupoint group （P〈0.05）;there was no significant difference between the acupuncture at non-acupoint group and the model group （P〉0.05）.Conclusion：Acupuncture may achieve the potential therapeutic effect on AD by regulating the structure and functional proteins of hippocampal mitochondria.展开更多
Proteins are essential parts of living organisms and participate in virtually every process within cells. As the genomlc sequences for increasing number of organisms are completed, research into how proteins can perfo...Proteins are essential parts of living organisms and participate in virtually every process within cells. As the genomlc sequences for increasing number of organisms are completed, research into how proteins can perform such a variety of functions has become much more intensive because the value of the genomic sequences relies on the accuracy of understanding the encoded gene products. Although the static three-dimensional structures of many proteins are known, the functions of proteins are ulti- mately governed by their dynamic characteristics, including the folding process, conformational fluctuations, molecular mo- tions, and protein-ligand interactions. In this review, the physicochemical principles underlying these dynamic processes are discussed in depth based on the free energy landscape （FEL） theory. Questions of why and how proteins fold into their native conformational states, why proteins are inherently dynamic, and how their dynamic personalities govern protein functions are answered. This paper will contribute to the understanding of structure-function relationship of proteins in the post-genome era of life science research.展开更多
Mollusc shell matrix proteins （SMPs） are important functional components embedded in the shell and play a role in shell formation. A SMP （Pif177） was identified previously from the nacreous layer of the Japanese p...Mollusc shell matrix proteins （SMPs） are important functional components embedded in the shell and play a role in shell formation. A SMP （Pif177） was identified previously from the nacreous layer of the Japanese pearl oyster Pinctadafucata, and its cleavage products （named pfPif97 and pfPif80 proteins） were found to bind to the chitin framework and induce aragonite crystal formation and orient the c axis. In this study, a homologue of pfPif177 was cloned from the mantle of the Pacific oyster Crassostrea gigas, containing the homologue of pfPif97 only and not pfPif80. This finding hints at the large divergence in gene structure between the two species. This homologue （cgPif97） shares characteristics with pfPif97, and suggests that the biological functions of these two proteins may be similar. The expression pattern of cgPif97 in different tissues and development stages in- dicates that it may play an important role in shell formation of the adult oyster. The morphology of the inner shell surface was af- fected by injected siRNA of cgPif97 and the calcite laths of the shell became thinner and narrower when the siRNA dose in- creased, suggesting that the cgPip7 gene plays an important role in calcite shell formation in C. gigas. In conclusion, we found evidence that the Pif177 gene evolved very fast but still retains a similar function among species [Current Zoology 59 （1）： 109-115, 2013].展开更多
One solid-state electrochemiluminescence （ECL） protein biosensor based on the competing reaction and substitute reaction between protein-to-DNA aptamer and DNA-to-DNA aptamer was proposed. Additionally, the biosenso...One solid-state electrochemiluminescence （ECL） protein biosensor based on the competing reaction and substitute reaction between protein-to-DNA aptamer and DNA-to-DNA aptamer was proposed. Additionally, the biosensor was based on ECL photo-quenching effect of ferrocene （Fc） to tris（2,2-bipyridyl）ruthenium（II） （Ru（bpy）2＋）. It was built up by modification of Au nanoparticles （AuNPs） and Ru（bpy）32＋ on one Au electrode firstly, and then self-assembly of one special double-stranded DNA （dsDNA） onto the electrode. This dsDNA was prepared by hybridization of one Fc labeled molecular beacon single-stranded DNA（ssDNA） and one anti-thrombin aptamer ssDNA. Without the target protein, this Fc-dsDNA/Ru（bpy）2＋- AuNPs/Au elec- trode trigged strong ECL signal, so we called it ECL ＂signal on＂ state. When thrombin was present in the sensing solution, the protein reacted with its aptamer from the Fc-dsDNA/Ru（bpy）3^2＋-AuNPs/Au electrode. Then the left molecular beacon ssDNA on the electrode recovered to its normal stem-loop structure and consequently its Fc labeler was close enough to the electrode surface to quench the ECL signal from Ru（bpy）3^2＋. It was in ECL ＂signal off＂ state. We measured the decrease in ECL intensity to sense the target protein. This was one endeavour to sense protein by using un-labeling target or probe strategy, which gave higher sensitivity and selectivity due to the better combination efficiency of protein and the un-labeled aptamer. 6.25 fmo/L thrombin was detected out,展开更多
Recognizing proteins via the production of highly specific monoclonal antibodies(mAbs)is crucial to identifying proteins for proteomic research.However,traditional mAb generation is time-consuming with low efficiency....Recognizing proteins via the production of highly specific monoclonal antibodies(mAbs)is crucial to identifying proteins for proteomic research.However,traditional mAb generation is time-consuming with low efficiency.In this study,we assessed the high throughput method of producing mAbs by immunizing mice with multiple antigens in order to obtain hybridomas against these multiple antigens in one cell fusion.We selected eight proteins that play important roles in human physiological or pathological processes.These proteins were mixed and simultaneously administered to one mouse.We observed the immunizing period for 10 d,and determined the effect of liquid medium and semi-solid medium in hybridoma generation.As a result,all eight immunogens induced antibodies in the immunized mouse in one cell fusion,we obtained hybridomas specific to all eight proteins by enzyme-linked immuno sorbent assay(ELISA)screening,hybridomas against five out of eight showed specific positive in Western-blotting assays.This indicates that we generated mAbs specific to eight proteins in one cell fusion,greatly increasing the efficiency of mAb generation.Furthermore,we observed that hybridomas selected from the liquid medium and semi-solid medium showed different reactivity to antigens.Our study established high-throughput and time-saving methods for production of mAbs.These results provide alternative approaches for increasing the efficacy of mAb generation.展开更多
基金Supported by The National Natural Science Foundation of China,No.30871147
文摘AIM:To assess BGC823 gastric cancer(GC) cell metastasis after knockdown of liver-intestine cadherin(CDH17) and the therapeutic value of CDH17-RNAilentivirus in vivo.METHODS:We evaluated primary tumor growth and assessed local infiltration and systemic tumor dissemination using an orthotopic implantation technique.The therapeutic value of CDH17 knockdown was examined by intratumoral administration of CDH17-RNA interference(RNAi)-lentivirus in an established GC tumor xenograft mouse model.Furthermore,a comparative proteomic approach was utilized to identify differentially expressed proteins in BGC823 and lenti-CDH17-miRneg cells following CDH17 knockdown.RESULTS:Metastases in the liver and lung appeared earlier and more frequently in animals with tumors derived from BGC823 or lenti-CDH17-miR-neg cells than in tumors derived from lenti-CDH17-miR-B cells.Average tumor weight and volume in the CDH17-RNAi-lentivirus-treated group were significantly lower than those in the control group(tumor volume:0.89 ± 0.04 cm 3 vs 1.16 ± 0.06 cm 3,P < 0.05;tumor weight:1.15 ± 0.58 g vs 2.09 ± 0.08 g,P < 0.05).Fifteen differentially expressed proteins were identified after CDH17 silencing in BGC823 cells,including a variety of cytoskeletal and chaperone proteins as well as proteins involved in metabolism,immunity/defense,cell proliferation and differentiation,cell cycle,and signal transduction.CONCLUSION:Our data establish a foundation for future studies of the comprehensive protein expression patterns and effects of CDH17 in GC.
文摘Adsorption reactions between surfaces of nanodiamond and nanosilica with diameter of 100 nm prepared as suspension solutions of 0.25μg/μL and lysozyme molecule with different concentrations of 7 mmol/L PPBS at pH=7, 9, 11, and 13 have been investigated by fluores- cence spectroscopy. Adsorption reaction constants and coverages of lysozyme with different concentrations of 0-1000 nmol/L under the influences of different pH values have been ob- tained. Helicities and conformations of the adsorbed lysozyme molecules, free spaces of every adsorbed lysozyme molecule on the surfaces of nanopartieles at different concentrations and pH values have been deduced and discussed. The highest adsorption capabilities for both sys- tems and conformational efficiency of the adsorbed lysozyme molecule at pH=13 have been obtained. Lysozyme molecules can be prepared, adsorbed and carried with optimal activity and helicity, with 2 and 10 mg/m2 on unit nanosurface, 130 and 150 mg/g with respect to the weight of nanoparticle, within the linear regions of the coverages at around 150-250 nmol/L and four pH values for nanodiamond and nanosilica, respectively. They can be prepared in the tightest packed form, with 20 and 55 mg/m2, 810-1680 and 580-1100 mg/g at threshold concentrations and four pH values for nanodiamond and nanosilica, respectively.
基金supported by the National Natural Science Foundation of China (21572043, 21473176)the Ministry of Science and Technology (2016YFA0400900, 2015CB910103)the Fundamental Research Funds for the Central Universities (PA2017GDQT0021)
文摘Snake toxin Calciseptine as a natural antagonist of L-type calcium channel has potential drug values, but its structural information remains unknown. Here, we report the total chemical synthesis of Calciseptine by using hydrazide based native chemical ligation. The crystal structure of Calciseptine was determined by racemic protein crystallography technique. Compared to the structure of its homologous family protein, we found that Calciseptine is adopting a typical three-finger structure.
基金The Ministry of Science and Technology of China(Grant No.2015CB559200)the National Natural Science Foundation of China(Grant No.81371432).
文摘Epilepsy is one of the most common neurological disorders characterized by epileptic seizures.The anti-epileptic drugs(AEDs)are the main form of treatment for people with epilepsy.Classically,people thought that AEDs modify the activities of ion channels to suppress epileptic seizures.However,accumulating evidence suggests that targeting at ion channels cannot completely account for the numerous effects of the AEDs on its broad clinical activity in epileptic patients.In our study,proteomic methods were used to quantify the proteome of hippocampal tissues from patients who were treated or not treated by AEDs(Carbamazepine).Further bioinformatics methods,including Gene Ontology(GO)annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis,were utilized to analyze the differences between two patient groups.We found that more than 400 proteins,including metabolism and immune related proteins,had a higher expression level in the carbamazepine-treated group compared with the controls.These altered proteins were considered to be involved in many different biological pathways.Among these pathways,immune-system related pathway modulated by complement C3 and microglia was highly remarkable,which regulated the synapses elimination in physiological condition.In epilepsy,the carbamazepine induced up-regulation of complement C3 might decrease the abnormal synaptic connections between neurons and thus contribute to the therapeutic role of carbamazepine.The results of our study suggested that apart from ion channels,carbamazepine exerted numerous effects on human epileptic foci,which might be the fundamental mechanisms of AEDs for treatment,adverse-effects and pharmacoresistance of epilepsy.
文摘Objective：To observe the effect of acupuncture on the expression of mitochondrial proteome in hippocampus of senescence-accelerated mouse prone g （SAMPg） mice models with Alzheimer disease （AD）,and to explore the possible protective mechanism of acupuncture on mitochondria.Methods：Sixty 6-month-old male SAMP8 mice were randomly divided into an acupuncture at acupoint group,an acupuncture at non-acupoint group and a model group,20 mice in each group.The 20 male senescence-accelerated mouse/resistance 1 （SAMR1） mice of the same age were used as a normal control group.Shenshu （BL 23）,Baihui （GV 20）,Xuehai （SP 10） and Geshu （BL 17） were selected for acupuncture intervention in acupuncture at acupoint group.After an 8-week intervention,mitochondrial tissues were extracted from the hippocampus.Differentially expressed proteins were identified by subcellular organelle proteomics.Western blot was used to verify the expressions of some related proteins in hippocampal mitochondria.Results：Compared with the model group,there were 13 differentially expressed protein spots in the acupuncture at acupoint group,of which,9 were up-regulated,including neurofilament light polypeptide （NFL）,actin （cytoplasmic 1,database ID：ACTB）,tubulin beta-2A chain （TBB2A）,tropomodulin-2 （TMOD2）,pyruvate dehydrogenase E1 component subunit beta （PDHE1-β）,NADH-ubiquinone oxidoreductase 75 kDa subunit （database ID：NDUS1）,heat shock cognate 71 kDa protein （HSC71）,pyruvate dehydrogenase E1 component subunit alpha （PDHE1-α） and ATP synthase beta subunit （ATP-β）;4 were down-regulated,including glial fibrillary acidic protein （GFAP）,pyruvate dehydrogenase phosphatase 1 （PDP1）,mitochondrial-processing peptidase subunit alpha （MMP-α） and adenosine kinase （ADK）.According to the information provided in the protein database,most of the differentially expressed proteins involve the regulation of mitochondrial function and structure.The expression levels of NFL and TBB2A in the normal control group and the acupuncture at acupoint group were significantly higher than those in the acupuncture at non-acupoint group （P〈0.05）.ATP-β and NDUS1 expression levels were significantly higher in the acupuncture at acupoint group than those in the acupuncture at non-acupoint group （P〈0.05）;there was no significant difference between the acupuncture at non-acupoint group and the model group （P〉0.05）.Conclusion：Acupuncture may achieve the potential therapeutic effect on AD by regulating the structure and functional proteins of hippocampal mitochondria.
基金supported by the National Natural Science Foundation of China(31370715,31160181,31360277,30860011)the National Basic Research Program of China(2013CB127500)+1 种基金the Program of Innovation Group of Yunnan Province(2011CI123)Foundation for Key Teacher in Yunnan University(XT412003)
文摘Proteins are essential parts of living organisms and participate in virtually every process within cells. As the genomlc sequences for increasing number of organisms are completed, research into how proteins can perform such a variety of functions has become much more intensive because the value of the genomic sequences relies on the accuracy of understanding the encoded gene products. Although the static three-dimensional structures of many proteins are known, the functions of proteins are ulti- mately governed by their dynamic characteristics, including the folding process, conformational fluctuations, molecular mo- tions, and protein-ligand interactions. In this review, the physicochemical principles underlying these dynamic processes are discussed in depth based on the free energy landscape （FEL） theory. Questions of why and how proteins fold into their native conformational states, why proteins are inherently dynamic, and how their dynamic personalities govern protein functions are answered. This paper will contribute to the understanding of structure-function relationship of proteins in the post-genome era of life science research.
文摘Mollusc shell matrix proteins （SMPs） are important functional components embedded in the shell and play a role in shell formation. A SMP （Pif177） was identified previously from the nacreous layer of the Japanese pearl oyster Pinctadafucata, and its cleavage products （named pfPif97 and pfPif80 proteins） were found to bind to the chitin framework and induce aragonite crystal formation and orient the c axis. In this study, a homologue of pfPif177 was cloned from the mantle of the Pacific oyster Crassostrea gigas, containing the homologue of pfPif97 only and not pfPif80. This finding hints at the large divergence in gene structure between the two species. This homologue （cgPif97） shares characteristics with pfPif97, and suggests that the biological functions of these two proteins may be similar. The expression pattern of cgPif97 in different tissues and development stages in- dicates that it may play an important role in shell formation of the adult oyster. The morphology of the inner shell surface was af- fected by injected siRNA of cgPif97 and the calcite laths of the shell became thinner and narrower when the siRNA dose in- creased, suggesting that the cgPip7 gene plays an important role in calcite shell formation in C. gigas. In conclusion, we found evidence that the Pif177 gene evolved very fast but still retains a similar function among species [Current Zoology 59 （1）： 109-115, 2013].
文摘One solid-state electrochemiluminescence （ECL） protein biosensor based on the competing reaction and substitute reaction between protein-to-DNA aptamer and DNA-to-DNA aptamer was proposed. Additionally, the biosensor was based on ECL photo-quenching effect of ferrocene （Fc） to tris（2,2-bipyridyl）ruthenium（II） （Ru（bpy）2＋）. It was built up by modification of Au nanoparticles （AuNPs） and Ru（bpy）32＋ on one Au electrode firstly, and then self-assembly of one special double-stranded DNA （dsDNA） onto the electrode. This dsDNA was prepared by hybridization of one Fc labeled molecular beacon single-stranded DNA（ssDNA） and one anti-thrombin aptamer ssDNA. Without the target protein, this Fc-dsDNA/Ru（bpy）2＋- AuNPs/Au elec- trode trigged strong ECL signal, so we called it ECL ＂signal on＂ state. When thrombin was present in the sensing solution, the protein reacted with its aptamer from the Fc-dsDNA/Ru（bpy）3^2＋-AuNPs/Au electrode. Then the left molecular beacon ssDNA on the electrode recovered to its normal stem-loop structure and consequently its Fc labeler was close enough to the electrode surface to quench the ECL signal from Ru（bpy）3^2＋. It was in ECL ＂signal off＂ state. We measured the decrease in ECL intensity to sense the target protein. This was one endeavour to sense protein by using un-labeling target or probe strategy, which gave higher sensitivity and selectivity due to the better combination efficiency of protein and the un-labeled aptamer. 6.25 fmo/L thrombin was detected out,
基金supported by the National Basic Research Program of China (2011CB915502)State Key Laboratory of Proteomics Project (SKLP Y200907)National High Technology Research and Development Program of China (2012AA020201)
文摘Recognizing proteins via the production of highly specific monoclonal antibodies(mAbs)is crucial to identifying proteins for proteomic research.However,traditional mAb generation is time-consuming with low efficiency.In this study,we assessed the high throughput method of producing mAbs by immunizing mice with multiple antigens in order to obtain hybridomas against these multiple antigens in one cell fusion.We selected eight proteins that play important roles in human physiological or pathological processes.These proteins were mixed and simultaneously administered to one mouse.We observed the immunizing period for 10 d,and determined the effect of liquid medium and semi-solid medium in hybridoma generation.As a result,all eight immunogens induced antibodies in the immunized mouse in one cell fusion,we obtained hybridomas specific to all eight proteins by enzyme-linked immuno sorbent assay(ELISA)screening,hybridomas against five out of eight showed specific positive in Western-blotting assays.This indicates that we generated mAbs specific to eight proteins in one cell fusion,greatly increasing the efficiency of mAb generation.Furthermore,we observed that hybridomas selected from the liquid medium and semi-solid medium showed different reactivity to antigens.Our study established high-throughput and time-saving methods for production of mAbs.These results provide alternative approaches for increasing the efficacy of mAb generation.
