[Objective] The aim of this study is to provide basis for developing genetic engineering vaccine and diagnostic kit for Theileria sergenti infection. [Objective] P23 gene of Theileria sergenti was amplified from its g...[Objective] The aim of this study is to provide basis for developing genetic engineering vaccine and diagnostic kit for Theileria sergenti infection. [Objective] P23 gene of Theileria sergenti was amplified from its genomic DNA by PCR amplification, and cloned into the pGEM-Easy vector; then the sequencing result was analyzed with bioinformatics methods. [Result] Whole length of the P23 gene from Theileria sergenti is 684 bp containing a 672 bp open reading frame. The deduced amino acid sequence (223 amino acid residues) contains a signal peptide of 19 amino acid residues and two fragments of transmembrane domains, with relative molecular weight of the 25.886 kD and with the pI of 9.22. The homology between the yielded sequence and Chitose of Theileria sergenti P23 gene(TS-Chitose type, D84446), Ikeda of Theileria sergenti P23 gene(TS-Ikeda type, D84447) reached 99% and 90%, respectively. The sequence has been accessed in GenBank(EU573168). [Conclusion] The protein encoded by the P23 gene has better stability and immunogenicity, thus can be used as the antigen candidate for preparing genetic engineering vaccine for Theileria sergenti.展开更多
Nuclear distribution gene C （NudC） was first found in Aspergillus nidulans as an upstream regulator of NudF, whose mamma- lian homolog is Lissencephaly 1 （Lisl）. NudC is conserved from fungi to mammals. Vertebrate...Nuclear distribution gene C （NudC） was first found in Aspergillus nidulans as an upstream regulator of NudF, whose mamma- lian homolog is Lissencephaly 1 （Lisl）. NudC is conserved from fungi to mammals. Vertebrate NudC has three homologs： NudC, NudC-like protein （NudCL）, and NudC-like protein 2 （NudCL2）. All members of the NudC family share a conserved p23 domain, which possesses chaperone activity both in conjunction with and independently of heat shock protein 90 （Hsp90）. Our group and the others found that NudC homologs were involved in cell cycle regulation by stabilizing the components of the LIS l/dynein complex. Additionally, NudC plays important roles in cell migration, ciliogenesis, thrombopoiesis, and the in- flammatory response. It has been reported that NudCL is essential for the stability of the dynein intermediate chain and cilio- genesis via its interaction with the dynein 2 complex. Our data showed that NudCL2 regulates the LISl/dynein pathway by stabilizing LIS 1 with Hsp90 chaperone. The fourth distantly related member of the NudC family, CML66, a tumor-associated antigen in human leukemia, contains a p23 domain and appears to promote oncogenesis by regulating the IGF-1R-MAPK sig- naling pathway. In this review, we summarize our current knowledge of the NudC family and highlight its potential clinical relevance.展开更多
[Objective] The aim was to study cloning and prokaryotic expression of P23 major surface protein gene of Theileria sergenti. [Method] A pair of specific primers was designed according to the sequence of P23 major surf...[Objective] The aim was to study cloning and prokaryotic expression of P23 major surface protein gene of Theileria sergenti. [Method] A pair of specific primers was designed according to the sequence of P23 major surface protein of T. sergenti (D84447).The P23 gene was amplified by PCR from genomic DNA of T. sergenti and cloned into pMD18-T vector to construct recombinant clonal vector pMD18-P23. Positive clones were identified by PCR screening and restriction digestion. A recombinant expression plasmid pGEX-4T-P23 was constructed by subcloning the cloned P23 gene into the linearized pGEX-4T-1 vector and transformed into E. coli BL21. After introduction by IPTG,the expressed fusion protein was identified by SDS-PAGE and Western-blotting. [Result] The cloned gene has a total length of 507 bp. Sequencing result showed that the nucleotide sequence of the cloned P23 gene shared 99.4% identity with that of P23 published in GenBank (D84447). The expressed fusion protein was 46 ku in molecular mass. Induction opportunity of zhours after culture inoculation was the best,the induction time of 6 h was the best,and induction temperature of 34 ℃ was the best as well,IPTG of 1 mmol/L had little effect on the expression. Western-blotting indicated that recombinant protein was recognized by specific antibody. [Conclusion] This study would lay a foundation for further research on the prevention and diagnose of T. sergenti.展开更多
A new ferritic creep resistant steel has been developed by eliminating Nb and adding 1.5 mass % Re to a ferritic steel grade T/P23 with the aim of enhancing its mechanical properties at high temperature.Cast ingots of...A new ferritic creep resistant steel has been developed by eliminating Nb and adding 1.5 mass % Re to a ferritic steel grade T/P23 with the aim of enhancing its mechanical properties at high temperature.Cast ingots of both steels, new grade and ASTM T/P 23, were hot rolled at 900℃ and then submitted to a thermal treatment consisting of solubilization at 1050℃ and tempering at 700℃. Tempered bainitic microstructures obtained contain second phases reinforcing carbide particles, mainly M_6C and M_（23）C_6 at the boundaries of both, prior austenite grains and bainitic ferrite laths, as well as MC within the grains. Mechanical properties at temperatures ranging from 540 to 600℃ were studied by strain-ratechange tests in compression at strain rates between 10^（-7） and 10^（-4）s^（-1）. These tests showed high stress exponents（n ≥ 20） and activation energies（Q ≈ 400 k J/mol） for both alloys, which were associated with a dislocation movement mechanism with a strong interaction between dislocations and precipitates. On the other hand, a creep exponent of 5 was derived for the stress dependence of minimum creep rate from conventional-type creep tests at 600℃. Although this stress exponent is usually related to a dislocation climb controlled creep mechanism, remarkable microstructural degradation observed with increasing creep time makes difficult to elucidate the true deformation mechanism controlling creep.展开更多
The cDNA of amphioxus p23, a highly conserved co-chaperone for Hsp90, was cloned into a bacterial expression vector pGEX - 6P - 1 and the GST-tagged fusion protein was produced in Eschherichia coli cells. The recombin...The cDNA of amphioxus p23, a highly conserved co-chaperone for Hsp90, was cloned into a bacterial expression vector pGEX - 6P - 1 and the GST-tagged fusion protein was produced in Eschherichia coli cells. The recombinant p23 was purified by affinity purification, and its molecular mass was estimated to be approximately 22 kDa by sodium dedecyl sulfate-polyacrylamide gel electrephoresis. The N-terminus of purified p23 was sequenced, and the resulting amino acid sequence matches exactly the predicted residues deduced from the amphioxus p23 gene. Besides, pelyclonal antibodies against the recombinant p23 were generated, and these antibodies not only recognized specifically the fusion protein GST - p23 from induced E. coli cells, purified GST - p23 and p23 protein, but also reacted with the total protein extracted fi＇om the adult amphioxus and formed a single positive band. These results pave the way for identifying its tissue and subcellular localization, and may open the door to clarifying its structure and mechanisms of biological role.展开更多
Inherited photoreceptor degeneration in humans constitutes a major cause of irreversible blindness in the world.They comprise various diseases,but retinitis pigmentosa is the most frequently observed.Retinitis pigment...Inherited photoreceptor degeneration in humans constitutes a major cause of irreversible blindness in the world.They comprise various diseases,but retinitis pigmentosa is the most frequently observed.Retinitis pigmentosa is commonly limited to the eye,where there is progressive photoreceptor degeneration,rods and secondarily cones.The mechanisms of cone and rod degeneration continue to be investigated,since most of the mutations causing retinitis pigmentosa affect rods and thus,the secondary death of cones is an intriguing question but,ultimately,the cause of blindness.Understanding the mechanisms of rod and cone degeneration could help us to develop therapies to stop or,at least,slow down the degeneration process.Secondary cone degeneration has been attributed to the trophic dependence between rods and cones,but microglial cell activation could also have a role.In this review,based on previous work carried out in our laboratory in early stages of photoreceptor degeneration in two animal models of retinitis pigmentosa,we show that microglial cell activation is observed prior to the the initiation of photoreceptor death.We also show that there is an increase of the retinal microglial cell densities and invasion of the outer retinal layers by microglial cells.The inhibition of the microglial cells improves photoreceptor survival and morphology,documenting a role for microglial cells in photoreceptor degeneration.Furthermore,these results indicate that the modulation of microglial cell reactivity can be used to prevent or diminish photoreceptor death in inherited photoreceptor degenerations.展开更多
文摘[Objective] The aim of this study is to provide basis for developing genetic engineering vaccine and diagnostic kit for Theileria sergenti infection. [Objective] P23 gene of Theileria sergenti was amplified from its genomic DNA by PCR amplification, and cloned into the pGEM-Easy vector; then the sequencing result was analyzed with bioinformatics methods. [Result] Whole length of the P23 gene from Theileria sergenti is 684 bp containing a 672 bp open reading frame. The deduced amino acid sequence (223 amino acid residues) contains a signal peptide of 19 amino acid residues and two fragments of transmembrane domains, with relative molecular weight of the 25.886 kD and with the pI of 9.22. The homology between the yielded sequence and Chitose of Theileria sergenti P23 gene(TS-Chitose type, D84446), Ikeda of Theileria sergenti P23 gene(TS-Ikeda type, D84447) reached 99% and 90%, respectively. The sequence has been accessed in GenBank(EU573168). [Conclusion] The protein encoded by the P23 gene has better stability and immunogenicity, thus can be used as the antigen candidate for preparing genetic engineering vaccine for Theileria sergenti.
基金supported by the Ministry of Science and Technology of China (2012CB945004, 2013CB945603)Natural Scientific Foundation of China (31125017, 31190063, 31100975, 31301149, 31471259)the 111 Project (B13026)
文摘Nuclear distribution gene C （NudC） was first found in Aspergillus nidulans as an upstream regulator of NudF, whose mamma- lian homolog is Lissencephaly 1 （Lisl）. NudC is conserved from fungi to mammals. Vertebrate NudC has three homologs： NudC, NudC-like protein （NudCL）, and NudC-like protein 2 （NudCL2）. All members of the NudC family share a conserved p23 domain, which possesses chaperone activity both in conjunction with and independently of heat shock protein 90 （Hsp90）. Our group and the others found that NudC homologs were involved in cell cycle regulation by stabilizing the components of the LIS l/dynein complex. Additionally, NudC plays important roles in cell migration, ciliogenesis, thrombopoiesis, and the in- flammatory response. It has been reported that NudCL is essential for the stability of the dynein intermediate chain and cilio- genesis via its interaction with the dynein 2 complex. Our data showed that NudCL2 regulates the LISl/dynein pathway by stabilizing LIS 1 with Hsp90 chaperone. The fourth distantly related member of the NudC family, CML66, a tumor-associated antigen in human leukemia, contains a p23 domain and appears to promote oncogenesis by regulating the IGF-1R-MAPK sig- naling pathway. In this review, we summarize our current knowledge of the NudC family and highlight its potential clinical relevance.
基金Supported by Science and Technology Development Program of Jilin Province (20050703-4)~~
文摘[Objective] The aim was to study cloning and prokaryotic expression of P23 major surface protein gene of Theileria sergenti. [Method] A pair of specific primers was designed according to the sequence of P23 major surface protein of T. sergenti (D84447).The P23 gene was amplified by PCR from genomic DNA of T. sergenti and cloned into pMD18-T vector to construct recombinant clonal vector pMD18-P23. Positive clones were identified by PCR screening and restriction digestion. A recombinant expression plasmid pGEX-4T-P23 was constructed by subcloning the cloned P23 gene into the linearized pGEX-4T-1 vector and transformed into E. coli BL21. After introduction by IPTG,the expressed fusion protein was identified by SDS-PAGE and Western-blotting. [Result] The cloned gene has a total length of 507 bp. Sequencing result showed that the nucleotide sequence of the cloned P23 gene shared 99.4% identity with that of P23 published in GenBank (D84447). The expressed fusion protein was 46 ku in molecular mass. Induction opportunity of zhours after culture inoculation was the best,the induction time of 6 h was the best,and induction temperature of 34 ℃ was the best as well,IPTG of 1 mmol/L had little effect on the expression. Western-blotting indicated that recombinant protein was recognized by specific antibody. [Conclusion] This study would lay a foundation for further research on the prevention and diagnose of T. sergenti.
基金supported by the Spanish Ministry of Economy and Competitiveness(MINECO)under Grant MAT2012-39124,MAT2015-68919,and MAT2016-80875
文摘A new ferritic creep resistant steel has been developed by eliminating Nb and adding 1.5 mass % Re to a ferritic steel grade T/P23 with the aim of enhancing its mechanical properties at high temperature.Cast ingots of both steels, new grade and ASTM T/P 23, were hot rolled at 900℃ and then submitted to a thermal treatment consisting of solubilization at 1050℃ and tempering at 700℃. Tempered bainitic microstructures obtained contain second phases reinforcing carbide particles, mainly M_6C and M_（23）C_6 at the boundaries of both, prior austenite grains and bainitic ferrite laths, as well as MC within the grains. Mechanical properties at temperatures ranging from 540 to 600℃ were studied by strain-ratechange tests in compression at strain rates between 10^（-7） and 10^（-4）s^（-1）. These tests showed high stress exponents（n ≥ 20） and activation energies（Q ≈ 400 k J/mol） for both alloys, which were associated with a dislocation movement mechanism with a strong interaction between dislocations and precipitates. On the other hand, a creep exponent of 5 was derived for the stress dependence of minimum creep rate from conventional-type creep tests at 600℃. Although this stress exponent is usually related to a dislocation climb controlled creep mechanism, remarkable microstructural degradation observed with increasing creep time makes difficult to elucidate the true deformation mechanism controlling creep.
文摘The cDNA of amphioxus p23, a highly conserved co-chaperone for Hsp90, was cloned into a bacterial expression vector pGEX - 6P - 1 and the GST-tagged fusion protein was produced in Eschherichia coli cells. The recombinant p23 was purified by affinity purification, and its molecular mass was estimated to be approximately 22 kDa by sodium dedecyl sulfate-polyacrylamide gel electrephoresis. The N-terminus of purified p23 was sequenced, and the resulting amino acid sequence matches exactly the predicted residues deduced from the amphioxus p23 gene. Besides, pelyclonal antibodies against the recombinant p23 were generated, and these antibodies not only recognized specifically the fusion protein GST - p23 from induced E. coli cells, purified GST - p23 and p23 protein, but also reacted with the total protein extracted fi＇om the adult amphioxus and formed a single positive band. These results pave the way for identifying its tissue and subcellular localization, and may open the door to clarifying its structure and mechanisms of biological role.
基金supported by grants from Fundación Séneca,Agencia de Cienciay Tecnología Región de Murcia,No.19881/GERM/15(to MVS)Spanish Ministry of Economy and Competitiveness,Instituto de Salud Carlos Ⅲ,Fondo Europeo de Desarrollo Regional ‘‘Una 30 Manera de Hacer Europa’’,No.SAF2015-67643-P(to MVS),PI16/00380(to MPVP),RD16/0008/0026(to MPVP),PI16/00031(to MAB)
文摘Inherited photoreceptor degeneration in humans constitutes a major cause of irreversible blindness in the world.They comprise various diseases,but retinitis pigmentosa is the most frequently observed.Retinitis pigmentosa is commonly limited to the eye,where there is progressive photoreceptor degeneration,rods and secondarily cones.The mechanisms of cone and rod degeneration continue to be investigated,since most of the mutations causing retinitis pigmentosa affect rods and thus,the secondary death of cones is an intriguing question but,ultimately,the cause of blindness.Understanding the mechanisms of rod and cone degeneration could help us to develop therapies to stop or,at least,slow down the degeneration process.Secondary cone degeneration has been attributed to the trophic dependence between rods and cones,but microglial cell activation could also have a role.In this review,based on previous work carried out in our laboratory in early stages of photoreceptor degeneration in two animal models of retinitis pigmentosa,we show that microglial cell activation is observed prior to the the initiation of photoreceptor death.We also show that there is an increase of the retinal microglial cell densities and invasion of the outer retinal layers by microglial cells.The inhibition of the microglial cells improves photoreceptor survival and morphology,documenting a role for microglial cells in photoreceptor degeneration.Furthermore,these results indicate that the modulation of microglial cell reactivity can be used to prevent or diminish photoreceptor death in inherited photoreceptor degenerations.
