Tay-Sachs disease and Sandhoff disease are severe hereditary neurodegenerative disorders caused by a deficiency ofβ-hexosaminidase A(HexA)enzyme,which results in the accumulation of GM2 gangliosides in the nervous sy...Tay-Sachs disease and Sandhoff disease are severe hereditary neurodegenerative disorders caused by a deficiency ofβ-hexosaminidase A(HexA)enzyme,which results in the accumulation of GM2 gangliosides in the nervous system cells.In this work,we analyzed the efficacy and safety of cell-mediated gene therapy for Sandhoff disease and Sandhoff disease using a bicistronic lentiviral vector encoding cDNA of HexAα-andβ-subunit genes separated by the nucleotide sequence of a P2A peptide(HEXA-HEXB).The functionality of the bicistronic construct containing the HEXA-HEXB genetic cassette was analyzed in a culture of HEK293T cells and human umbilical cord blood mononuclear cells(hUCBMCs).Our results showed that the enzymatic activity of HexA in the conditioned medium harvested from genetically modified HEK293T-HEXA-HEXB and hUCBMCs-HEXA-HEXB was increased by 23 and 8 times,respectively,compared with the conditioned medium of native cells.Western blot analysis showed that hUCBMCs-HEXA-HEXB secreted both completely separated HEXA and HEXB proteins,and an uncleaved protein containing HEXA+HEXB linked by the P2A peptide.Intravenous injection of genetically modified hUCBMCs-HEXA-HEXB to laboratory Wistar rats was carried out,and the HexA enzymatic activity in the blood plasma of experimental animals,as well as the number of live cells of immune system organs(spleen,thymus,bone marrow,lymph nodes)were determined.A significant increase in the enzymatic activity of HexA in the blood plasma of laboratory rats on days 6 and 9(by 2.5 and 3 times,respectively)after the administration of hUCBMCsHEXA-HEXB was shown.At the same time,the number of live cells in the studied organs remained unchanged.Thus,the functionality of the bicistronic genetic construct encoding cDNA of the HEXA and HEXB genes separated by the nucleotide sequence of the P2A peptide was shown in vitro and in vivo.We hypothesize that due to the natural ability of hUCBMCs to overcome biological barriers,such a strategy can restore the activity of the missing enzyme in the central nervous system of patients with GM2 gangliosidoses.Based on the obtained data,it can be concluded that intravenous administration of hUCBMCs with HexA overexpression is a promising method of the therapy for GM2 gangliosidoses.The animal protocol was approved by the Animal Ethics Committee of the Kazan Federal University(No.23)on June 30,2020.展开更多
Many animal feed grains contain high β-glucan in the cell wall. Pigs do not secret β-glucanase to degrade the β-glucan in their feed. The indigestible β-glucan not only blocks the release of nutrients from the gra...Many animal feed grains contain high β-glucan in the cell wall. Pigs do not secret β-glucanase to degrade the β-glucan in their feed. The indigestible β-glucan not only blocks the release of nutrients from the grain cell wall, but also increases the digesta viscosity in the gastrointestinal tract of pigs. Therefore, dietary β-glucan significantly inhibits nutrient digestion and absorption in pigs. Transgenic expression of β-glucanase in the digestive tract of pigs may offer a solution to solve this problem. In the current study, four artificial codon-optimized β-glucanases genes was prepared and expressed in porcine cells. Only p Bg A and p Egx showed high activity in transfected pig kidney cells. To improve the p H range and p H stability of β-glucanase, the two β-glucanases, p Bg A and p Egx, were co-expressed in pig kidney cells and salivary gland cells by Linker A3 or 2A peptide. The resulting dual enzymes of p Bg A3 p Eg and p Bg2 Ap Eg showed significantly enlarged p H range and significantly increased p H stability, as compared to parental enzymes. These results provide useful data for future study on increasing the feed digestibility of pigs by transgenic expression of β-glucanase in their salivary glands.展开更多
Chemokines are cytokines that can promote the activation and migration of immune cells,and increase the recognition of antigen by antigen-presenting cells(APC).Previous studies showed that a DNA vaccine can induce hum...Chemokines are cytokines that can promote the activation and migration of immune cells,and increase the recognition of antigen by antigen-presenting cells(APC).Previous studies showed that a DNA vaccine can induce humoral and cellular immune responses of flounder after immunization.To explore the improvement of chemokines on the efficiency of OmpK vaccine,two bicistronic DNA candidate vaccines were constructed and the immune responses they induced in the flounder were investigated by reverse transcription polymerase chain reaction(RT-PCR),indirect immunofl uorescent assay(IFA),H&E staining,fl ow cytometry(FCM),and quantifi cational real-time polymerase chain reaction(qRT-PCR).pBudCE4.1 plasmid as an expression vector,bicistronic DNA vaccines encoding OmpK gene and CC-motif ligand 4 gene(p-OmpK-CCL4),or Ompk gene and CC-motif ligand 19 gene(p-OmpK-CCL19)were successfully constructed.The results showed that two bicistronic DNA vaccines expressed Ompk protein of Vibrio anguillarum and CCL4/CCL19 proteins of fl ounder both in vitro and in vivo.After immunization,a large number of leucocytes in muscle were recruited at the injection site in treatment groups.The constructed vaccines induced signifi cant increases in CD4-1^(+) and CD4-2^(+) T lymphocytes,and sIgM^(+) B lymphocytes in peripheral blood,spleen,and head kidney.The percentage of T lymphocytes peaked on the 14^(th) post-vaccination day whereas that of B lymphocytes peaked in the 6^(th) post-vaccination week.Moreover,the expression profi les of 10 immune-related genes increased in muscles around the injection site,spleen,and head kidney.After the challenge,p-OmpK-CCL4 and p-OmpK-CCL19 conferred a relative percentage survival(RPS)of 74.1%and 63.3%,respectively,higher than p-OmpK alone(40.8%).In conclusion,both CCL4 and CCL19 can improve the protection of p-OmpK via evoking local immune response and then humoral and cellular immunity.CCL4 and CCL19 will be potential molecular adjuvants for use in DNA vaccines.展开更多
Objective: To construct bicistronic expression vector with RANTES and SDF-1 genes, the ligands of HIV-1 principal coreceptors, and identify its expression. Methods: RANTES-KDEL was amplified from plasmid pCMV-R-K by P...Objective: To construct bicistronic expression vector with RANTES and SDF-1 genes, the ligands of HIV-1 principal coreceptors, and identify its expression. Methods: RANTES-KDEL was amplified from plasmid pCMV-R-K by PCR and cloned into eukaryotic expression vector pCMV-S/K. Gene transfection into HeLa cells was carried out by lipofectin. Indirect immumofluorescence and radioimmunoprecipitation were used to confirm the expression of RANTES and SDF-1. Results: The construction of pCMV-R-K-S-K was confirmed by enzymatic digestion and sequencing. RANTES and SDF-1 were shown expressed in HeLa cells by indirect immumofluorescence and radioimmunoprecipitation. Conclusion: pCMV-R-K-S-K was constructed and expressed in cell line Hela successfully, which will contribute to further study of gene therapy of AIDS by HIV-1 coreceptors knockout.展开更多
Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by...Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase展开更多
It has been well documented that bone morphogenetic .proteins （BMPs）, a group of proteins belonging to the transforming growth factor-β （TGFβ） superfamily, can induce bone formation, both in vivo and in vitro. B...It has been well documented that bone morphogenetic .proteins （BMPs）, a group of proteins belonging to the transforming growth factor-β （TGFβ） superfamily, can induce bone formation, both in vivo and in vitro. Bone morphogenetic protein-2 （BMP2） is a potent osteoinductive factor and is being evaluated as a bone growth inducer for orthopedic applications.1 Vascular endothelial growth factor （VEGF）, the best-characterized angiogenic factor,展开更多
In the present study, follistatin(FST) gene expression vectors with either a bicistronic gene transfer cassette alone, or a bicistron gene cassette carrying a matrix attachment region(MAR) were constructed and transfe...In the present study, follistatin(FST) gene expression vectors with either a bicistronic gene transfer cassette alone, or a bicistron gene cassette carrying a matrix attachment region(MAR) were constructed and transfected to bovine fetal fibroblasts. Evaluations of both the integration and expression of exogenous FST indicated that the p MAR-CAG-FST-IRES-Ac GFP1-poly A-MAR(pMAR-FST) vector had higher capacity to form monoclonal transgenic cells than the vector without MAR,though transient transfection and integration efficiency were similar with either construct. Remarkably, protein expression in transgenic cells with the p MAR-FST vector was significantly higher than that from the bicistronic vector. Exogenous FST was expressed in all of the p MARFST transgenic mice at F_0, F_1 and F_2. Total muscle growth in F_0 mice was significantly greater than in wild-type mice,with larger muscles in fore and hind limbs of transgenic mice. p MAR-FST transgenic mice were also found with more evenly distributed muscle bundles and thinner spaces between sarcolemma, which suggests a correlation between transgene expression-associated muscle development and the trend of muscle growth. In conclusion, a p MAR-FST vector, which excluded the resistant genes and frame structure, enhances and stabilizes FST gene expressions in both transfected cells and transgenic mice.展开更多
Most eukaryotic mRNAs are translated in a cap-dependent fashion;however,under stress conditions,the cap-independent translation driven by internal ribosomal entry sites(IRESs)can serve as an alternative mechanism for ...Most eukaryotic mRNAs are translated in a cap-dependent fashion;however,under stress conditions,the cap-independent translation driven by internal ribosomal entry sites(IRESs)can serve as an alternative mechanism for protein production.Many IRESs have been discovered from viral or cellular mRNAs to promote ribosome assembly and initiate translation by recruiting different trons-acting factors.Although the mechanisms of translation initiation driven by viral IRESs are relatively well understood,the existence of cellular IRESs is still under debate due to the limitations of translation reporter systems used to assay IRES activities.A recent screen identified>1000 putative IRESs from viral and human mRNAs,expanding the scope and mechanism for capindependent translation.Additionally,a large number of circular RNAs lacking free ends were identified in eukaryotic cells,many of which are found to be translated through IRESs.These findings suggest that IRESs may play a previously unappreciated role in driving translation of the new type of mRNA,implying a hidden proteome produced from cap-independent translation.展开更多
基金funded by the subsidy allocated to Kazan Federal University for the state assignment#0671-2020-0058 in the sphere of scientific activities。
文摘Tay-Sachs disease and Sandhoff disease are severe hereditary neurodegenerative disorders caused by a deficiency ofβ-hexosaminidase A(HexA)enzyme,which results in the accumulation of GM2 gangliosides in the nervous system cells.In this work,we analyzed the efficacy and safety of cell-mediated gene therapy for Sandhoff disease and Sandhoff disease using a bicistronic lentiviral vector encoding cDNA of HexAα-andβ-subunit genes separated by the nucleotide sequence of a P2A peptide(HEXA-HEXB).The functionality of the bicistronic construct containing the HEXA-HEXB genetic cassette was analyzed in a culture of HEK293T cells and human umbilical cord blood mononuclear cells(hUCBMCs).Our results showed that the enzymatic activity of HexA in the conditioned medium harvested from genetically modified HEK293T-HEXA-HEXB and hUCBMCs-HEXA-HEXB was increased by 23 and 8 times,respectively,compared with the conditioned medium of native cells.Western blot analysis showed that hUCBMCs-HEXA-HEXB secreted both completely separated HEXA and HEXB proteins,and an uncleaved protein containing HEXA+HEXB linked by the P2A peptide.Intravenous injection of genetically modified hUCBMCs-HEXA-HEXB to laboratory Wistar rats was carried out,and the HexA enzymatic activity in the blood plasma of experimental animals,as well as the number of live cells of immune system organs(spleen,thymus,bone marrow,lymph nodes)were determined.A significant increase in the enzymatic activity of HexA in the blood plasma of laboratory rats on days 6 and 9(by 2.5 and 3 times,respectively)after the administration of hUCBMCsHEXA-HEXB was shown.At the same time,the number of live cells in the studied organs remained unchanged.Thus,the functionality of the bicistronic genetic construct encoding cDNA of the HEXA and HEXB genes separated by the nucleotide sequence of the P2A peptide was shown in vitro and in vivo.We hypothesize that due to the natural ability of hUCBMCs to overcome biological barriers,such a strategy can restore the activity of the missing enzyme in the central nervous system of patients with GM2 gangliosidoses.Based on the obtained data,it can be concluded that intravenous administration of hUCBMCs with HexA overexpression is a promising method of the therapy for GM2 gangliosidoses.The animal protocol was approved by the Animal Ethics Committee of the Kazan Federal University(No.23)on June 30,2020.
基金funded by a grant from the National Science and Technology Major Projects of China (2014ZX08006004)three grants from the Department of Science and Technology of Guangdong,China (20111090700016,2011A020102003 and 2011A020201009)
文摘Many animal feed grains contain high β-glucan in the cell wall. Pigs do not secret β-glucanase to degrade the β-glucan in their feed. The indigestible β-glucan not only blocks the release of nutrients from the grain cell wall, but also increases the digesta viscosity in the gastrointestinal tract of pigs. Therefore, dietary β-glucan significantly inhibits nutrient digestion and absorption in pigs. Transgenic expression of β-glucanase in the digestive tract of pigs may offer a solution to solve this problem. In the current study, four artificial codon-optimized β-glucanases genes was prepared and expressed in porcine cells. Only p Bg A and p Egx showed high activity in transfected pig kidney cells. To improve the p H range and p H stability of β-glucanase, the two β-glucanases, p Bg A and p Egx, were co-expressed in pig kidney cells and salivary gland cells by Linker A3 or 2A peptide. The resulting dual enzymes of p Bg A3 p Eg and p Bg2 Ap Eg showed significantly enlarged p H range and significantly increased p H stability, as compared to parental enzymes. These results provide useful data for future study on increasing the feed digestibility of pigs by transgenic expression of β-glucanase in their salivary glands.
基金Supported by the National Natural Science Foundation of China(Nos.32173005,31730101,31672684)the National Key Research and Development Program of China(No.2018YFD0900503)+5 种基金the Shandong Provincial Natural Science Foundation(No.ZR2020KC025)the Fundamental Research Funds for the Central Universities(No.201822015)the Director Foundation of Functional Laboratory for Marine Fisheries Science and Food Production Processes,Qingdao National Laboratory for Marine Science and Technology(No.2018MFSD-01)the NBRPC(No.2012CB114406)the Key Research and Development Program of Shandong Province(No.2016GNC115001)the Taishan Scholar Program of Shandong Province。
文摘Chemokines are cytokines that can promote the activation and migration of immune cells,and increase the recognition of antigen by antigen-presenting cells(APC).Previous studies showed that a DNA vaccine can induce humoral and cellular immune responses of flounder after immunization.To explore the improvement of chemokines on the efficiency of OmpK vaccine,two bicistronic DNA candidate vaccines were constructed and the immune responses they induced in the flounder were investigated by reverse transcription polymerase chain reaction(RT-PCR),indirect immunofl uorescent assay(IFA),H&E staining,fl ow cytometry(FCM),and quantifi cational real-time polymerase chain reaction(qRT-PCR).pBudCE4.1 plasmid as an expression vector,bicistronic DNA vaccines encoding OmpK gene and CC-motif ligand 4 gene(p-OmpK-CCL4),or Ompk gene and CC-motif ligand 19 gene(p-OmpK-CCL19)were successfully constructed.The results showed that two bicistronic DNA vaccines expressed Ompk protein of Vibrio anguillarum and CCL4/CCL19 proteins of fl ounder both in vitro and in vivo.After immunization,a large number of leucocytes in muscle were recruited at the injection site in treatment groups.The constructed vaccines induced signifi cant increases in CD4-1^(+) and CD4-2^(+) T lymphocytes,and sIgM^(+) B lymphocytes in peripheral blood,spleen,and head kidney.The percentage of T lymphocytes peaked on the 14^(th) post-vaccination day whereas that of B lymphocytes peaked in the 6^(th) post-vaccination week.Moreover,the expression profi les of 10 immune-related genes increased in muscles around the injection site,spleen,and head kidney.After the challenge,p-OmpK-CCL4 and p-OmpK-CCL19 conferred a relative percentage survival(RPS)of 74.1%and 63.3%,respectively,higher than p-OmpK alone(40.8%).In conclusion,both CCL4 and CCL19 can improve the protection of p-OmpK via evoking local immune response and then humoral and cellular immunity.CCL4 and CCL19 will be potential molecular adjuvants for use in DNA vaccines.
文摘Objective: To construct bicistronic expression vector with RANTES and SDF-1 genes, the ligands of HIV-1 principal coreceptors, and identify its expression. Methods: RANTES-KDEL was amplified from plasmid pCMV-R-K by PCR and cloned into eukaryotic expression vector pCMV-S/K. Gene transfection into HeLa cells was carried out by lipofectin. Indirect immumofluorescence and radioimmunoprecipitation were used to confirm the expression of RANTES and SDF-1. Results: The construction of pCMV-R-K-S-K was confirmed by enzymatic digestion and sequencing. RANTES and SDF-1 were shown expressed in HeLa cells by indirect immumofluorescence and radioimmunoprecipitation. Conclusion: pCMV-R-K-S-K was constructed and expressed in cell line Hela successfully, which will contribute to further study of gene therapy of AIDS by HIV-1 coreceptors knockout.
文摘Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase
文摘It has been well documented that bone morphogenetic .proteins （BMPs）, a group of proteins belonging to the transforming growth factor-β （TGFβ） superfamily, can induce bone formation, both in vivo and in vitro. Bone morphogenetic protein-2 （BMP2） is a potent osteoinductive factor and is being evaluated as a bone growth inducer for orthopedic applications.1 Vascular endothelial growth factor （VEGF）, the best-characterized angiogenic factor,
基金supported by the National Transgenic Breeding Project (2014ZX08007-002)
文摘In the present study, follistatin(FST) gene expression vectors with either a bicistronic gene transfer cassette alone, or a bicistron gene cassette carrying a matrix attachment region(MAR) were constructed and transfected to bovine fetal fibroblasts. Evaluations of both the integration and expression of exogenous FST indicated that the p MAR-CAG-FST-IRES-Ac GFP1-poly A-MAR(pMAR-FST) vector had higher capacity to form monoclonal transgenic cells than the vector without MAR,though transient transfection and integration efficiency were similar with either construct. Remarkably, protein expression in transgenic cells with the p MAR-FST vector was significantly higher than that from the bicistronic vector. Exogenous FST was expressed in all of the p MARFST transgenic mice at F_0, F_1 and F_2. Total muscle growth in F_0 mice was significantly greater than in wild-type mice,with larger muscles in fore and hind limbs of transgenic mice. p MAR-FST transgenic mice were also found with more evenly distributed muscle bundles and thinner spaces between sarcolemma, which suggests a correlation between transgene expression-associated muscle development and the trend of muscle growth. In conclusion, a p MAR-FST vector, which excluded the resistant genes and frame structure, enhances and stabilizes FST gene expressions in both transfected cells and transgenic mice.
基金This work is supported by the National Natural Science Foundation of China(31570823,31661143031,and 31730110 to Z.W.,91753135 and 31870814 to Y.Y.)Z.W.is also supported by the CAS Pioneer 100-Talent Program(type A).Y.Y.is also sponsored by the Youth Innovation Promotion Association CAS and Shanghai Scienee and Technology Committee Rising-Star Program(19QA1410500).
文摘Most eukaryotic mRNAs are translated in a cap-dependent fashion;however,under stress conditions,the cap-independent translation driven by internal ribosomal entry sites(IRESs)can serve as an alternative mechanism for protein production.Many IRESs have been discovered from viral or cellular mRNAs to promote ribosome assembly and initiate translation by recruiting different trons-acting factors.Although the mechanisms of translation initiation driven by viral IRESs are relatively well understood,the existence of cellular IRESs is still under debate due to the limitations of translation reporter systems used to assay IRES activities.A recent screen identified>1000 putative IRESs from viral and human mRNAs,expanding the scope and mechanism for capindependent translation.Additionally,a large number of circular RNAs lacking free ends were identified in eukaryotic cells,many of which are found to be translated through IRESs.These findings suggest that IRESs may play a previously unappreciated role in driving translation of the new type of mRNA,implying a hidden proteome produced from cap-independent translation.
