The outbreak of coronavirus disease 2019 has seriously threatened human health.Rapidly and sensitively detecting SARSCoV-2 viruses can help control the spread of viruses.However,it is an arduous challenge to apply sem...The outbreak of coronavirus disease 2019 has seriously threatened human health.Rapidly and sensitively detecting SARSCoV-2 viruses can help control the spread of viruses.However,it is an arduous challenge to apply semiconductor-based substrates for virus SERS detection due to their poor sensitivity.Therefore,it is worthwhile to search novel semiconductor-based substrates with excellent SERS sensitivity.Herein we report,for the first time,Nb2C and Ta2C MXenes exhibit a remarkable SERS enhancement,which is synergistically enabled by the charge transfer resonance enhancement and electromagnetic enhancement.Their SERS sensitivity is optimized to 3.0×10^6 and 1.4×10^6 under the optimal resonance excitation wavelength of 532 nm.Additionally,remarkable SERS sensitivity endows Ta2C MXenes with capability to sensitively detect and accurately identify the SARS-CoV-2 spike protein.Moreover,its detection limit is as low as 5×10^−9 M,which is beneficial to achieve real-time monitoring and early warning of novel coronavirus.This research not only provides helpful theoretical guidance for exploring other novel SERS-active semiconductor-based materials but also provides a potential candidate for the practical applications of SERS technology.展开更多
AIM: To investigate the transactivating effect of complete S protein of hepatitis B virus (HBV) and to construct a subtractive cDNA library of genes transactivated by complete S protein of HBV by suppression subtracti...AIM: To investigate the transactivating effect of complete S protein of hepatitis B virus (HBV) and to construct a subtractive cDNA library of genes transactivated by complete S protein of HBV by suppression subtractive hybridization (SSH) technique and to clone genes associated with its transactivation activity, and to pave the way for elucidating the pathogenesis of hepatitis B virus infection. METHODS: pcDNA3.1(-)-complete S containing full-length HBV S gene was constructed by insertion of HBV complete S gene into BamH I/Kpn I sites. HepG2 cells were cotransfected with pcDNA3.1(-)-complete S and pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-galactosidase (β-gal). Suppression subtractive hybridization and bioinformatics techniques were used. The mRNA of HepG2 cells transfected with pcDNA3.Incomplete S and pcDNA3.1(-) empty vector was isolated, and detected for the expression of complete S protein by reverse transcription polymerase chain reaction (RT-PCR) method, and cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptors 1 and 2, respectively. After tester cDNA had been hybridized with driver cDNA twice and underwent nested PCR twice, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out within E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR) amplification. RESULTS: The complete S mRNA could be detected by RT-PCR in HepG2 cells transfected with the pcDNA3.1(-)-complete S. The activity of β-gal in HepG2 cells transfected with the pcDNA3.1(-)-complete s was 6.9 times higher than that of control plasmid. The subtractive library of genes transactivated by HBV complete S protein was constructed successfully. The amplified library contains 86 positive clones. Colony PCR showed that 86 clones contained DNA inserts of 200-1 000 bp, respectively. Sequence analysis was performed in 35 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 33 coding sequences were obtained. These cDNA sequences might be target genes transactivated by complete S protein of HBV. Moreover, two unknown genes were discovered, full length coding sequences were obtained by bioinformatics techniques, one of them was named complete S transactivated protein 1 (CSTP1) and registered in GenBank (AY553877). CONCLUSION: The complete S gene of HBV has a transactivating effect on SV40 early promoter. A subtractive cDNA library of genes transactivated by HBV complete S protein using SSH technique has been constructed successfully. The obtained sequences may be target genes transactivated by HBV complete S protein among which some genes coding proteins are involved in cell cycle regulation, metabolism, immunity, signal transduction, cell apoptosis and formation mechanism of hepatic carcinoma.展开更多
[Objective] To obtain pure recombinant S1 and S2 of SARS S protein. [Method] Using asymmetric PCR and ligation with endonuclease, S1 and S2 fragments of SARSV HK strain S gene were synthesized. Then, these two fragmen...[Objective] To obtain pure recombinant S1 and S2 of SARS S protein. [Method] Using asymmetric PCR and ligation with endonuclease, S1 and S2 fragments of SARSV HK strain S gene were synthesized. Then, these two fragments were inserted into plasmid pET28a to obtain recombinant vectors pET28a-S1 and pET28a-S2, respectively. These recombinant vectors were transformed into E. coli BL21, and expression of S1 and S2 fragments were induced by IPTG. The conditions of expression and purification were optimized. [Result] The S1 and S2 fragments were amplified and successfully expressed in E. coli. [Conclusion] This research provides detection antigens for follow-up development of SARS vaccine.展开更多
AIM: To investigate the biological function of complete S protein and to look for proteins interacting with complete S protein in hepatocytes. METHODS: We constructed bait plasmid expressing complete S protein of HBV ...AIM: To investigate the biological function of complete S protein and to look for proteins interacting with complete S protein in hepatocytes. METHODS: We constructed bait plasmid expressing complete S protein of HBV by cloning the gene of complete S protein into pGBKT7, then the recombinant plasmid DNA was transformed into yeast AH109 (a type). The transformed yeast AH109 was mated with yeast Y187 (α type) containing liver cDNA library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-α-gal for selection and screening. After extracting and sequencing of plasmids from positive (blue) colonies, we underwent sequence analysis by bioinformatics. RESULTS: Nineteen colonies were selected and sequenced. Among them, five colonies were Homo sapiens solute carrier family 25, member 23 (SLC25A23), one was Homo sapiens calrer.iculin, one was human serum albumin (ALB) gene, one was Homo sapiens metallothionein 2A, two were Homo sapiens betaine-homocysteine methyltransferase, three were Homo sapiens Na+ and H+coupled amino acid transport system N, one was Homo sapiens CD81 antigen (target of anti-proliferative antibody 1) (CD81), three were Homo sapiens diazepam binding inhibitor, two colonies were new genes with unknown function. CONCLUSION: The yeast-two hybrid system is an effective method for identifying hepatocyte proteins interacting with complete S protein of HBV. The complete S protein may bind to different proteins i.e., its multiple functions in vivo.展开更多
Porcine epidemic diarrhea(PED)caused by the porcine epidemic diarrhea virus(PEDV),is a severe infectious and devastating swine disease that leads to serious economic losses in the swine industry worldwide.An increased...Porcine epidemic diarrhea(PED)caused by the porcine epidemic diarrhea virus(PEDV),is a severe infectious and devastating swine disease that leads to serious economic losses in the swine industry worldwide.An increased number of PED cases caused by variant PEDV have been reported in many countries since 2010.S protein is the main immunogenic protein containing some B-cell epitopes that can induce neutralizing antibodies of PEDV.In this study,the construction,expression and purification of Pseudomonas aeruginosa exotoxin A(PE)without domain Ⅲ(PE△Ⅲ)as a vector was performed for the delivery of PEDV S-A or S-B.PE(△Ⅲ)PEDV S-A and PE(△Ⅲ)PEDV S-B recombinant proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis.The immunogenicity of PEDV S-A and PEDV S-B subunit vaccines were evaluated in mice.The results showed that PEDV-S-B vaccine could not only induce specific humoral and Th1 type-dominant cellular immune responses,but also stimulate PEDV-specific mucosal immune responses in mice.PEDV-S-B subunit vaccine is a novel candidate mucosal vaccine against PEDV infection.展开更多
C/EBP homologous protein, an important transcription factor during endoplasmic reticulum stress, participates in cell apoptosis mediated by endoplasmic reticulum stress. Previous studies have shown that C/EBP homologo...C/EBP homologous protein, an important transcription factor during endoplasmic reticulum stress, participates in cell apoptosis mediated by endoplasmic reticulum stress. Previous studies have shown that C/EBP homologous protein mediates nerve injury during Alzheimer＇s disease, subarachnoid hemorrhage and spinal cord trauma. In this study, we introduced C/EBP homologous protein short hairpin RNA into the brains of ischemia/reperfusion rat models via injection of lentiviral vector through the left lateral ventricle. Silencing C/EBP homologous protein gene expression significantly reduced cerebral infarction volume, decreased water content and tumor necrosis factor-α and interleukin-1β mRNA expression in brain tissues following infarction, diminished the number of TUNEL-positive cells in the infarct region, decreased caspase-3 protein content and increased Bcl-2 protein content. These results suggest that silencing C/EBP homologous protein lessens cell apoptosis and inflammatory reactions, thereby protecting nerves.展开更多
Meanwhile the outbreak of the Covid-19 since December, 2019 in China, it has killed more than a hundred thousand of people of all ages and sex across the globe in a short span of time. On the bases of this study the n...Meanwhile the outbreak of the Covid-19 since December, 2019 in China, it has killed more than a hundred thousand of people of all ages and sex across the globe in a short span of time. On the bases of this study the nearest family member of the virus and its receptor binding domain of S protein including its model structure and function of its active sites were naked through Multiple Sequence Alignment, modelling and molecular docking software accordingly its repository genome databases. The virus was genetically associated and molecular evolutionary related with (<em>RaTG</em>13) and it scores 96.12% homology with 99% query coverage followed by <em>bat-SL-CoVZC</em>45 and<em> bat-SL-CoVZXC</em>21 notch 89.12% and 88.65% respectively. However, SARS and MERS corona type virus those outbreak earlier respectively less likely family members of 2019-nCoV. Though the virus has a close genetic association with those previous SARS coronaviruses, and certainly the spike protein used as a binding receptor to fight against human receptor protein of ACE 2, but on the basis of FRODOC and HDOCK server analysis multi favorable active sites of S protein was discovered such GLN493 shown as a finest key in both model and possessed a unique traits on it resulting unexpected rate of transmission and number of people died while compared to the previous one. TYR500, ASN501, GLN498 and others residues preferably contemplate site also. In particular, the diversity of the virus in the world may be due to the genome structure of the virus and S gene changed over the time, across the world against to host of human genetic diversity, which may be more robust, and may be a new and unique feature. This is because it is characterized close to contact with distance divergence between wild type novel coronavirus which was risen from China against to the genomes from Lebanon, India, Italy, and USA and so on. Thus, the World Health Organization and its researchers should focus on immunologic research and effective drug and vaccine development that will help to address the epidemiology of the virus, which can provide a long-term solution.展开更多
基金The authors gratefully acknowledge the finical support of the National Key Research and Development Project(No.2017YFB0310600)this work is also supported by Shanghai International Science and Technology Cooperation Fund(Nos.17520711700 and 18520744200).
文摘The outbreak of coronavirus disease 2019 has seriously threatened human health.Rapidly and sensitively detecting SARSCoV-2 viruses can help control the spread of viruses.However,it is an arduous challenge to apply semiconductor-based substrates for virus SERS detection due to their poor sensitivity.Therefore,it is worthwhile to search novel semiconductor-based substrates with excellent SERS sensitivity.Herein we report,for the first time,Nb2C and Ta2C MXenes exhibit a remarkable SERS enhancement,which is synergistically enabled by the charge transfer resonance enhancement and electromagnetic enhancement.Their SERS sensitivity is optimized to 3.0×10^6 and 1.4×10^6 under the optimal resonance excitation wavelength of 532 nm.Additionally,remarkable SERS sensitivity endows Ta2C MXenes with capability to sensitively detect and accurately identify the SARS-CoV-2 spike protein.Moreover,its detection limit is as low as 5×10^−9 M,which is beneficial to achieve real-time monitoring and early warning of novel coronavirus.This research not only provides helpful theoretical guidance for exploring other novel SERS-active semiconductor-based materials but also provides a potential candidate for the practical applications of SERS technology.
基金Supported by the National Natural Science Foundation of China, No. C03011402, No. C30070690 the Science and Technique Foundation of PLA during the 9th Five-year Plan period, No. 98D063the Launching Foundation for Students Studying Abroad of PLA, No. 98H038the Youth Science and Technique Foundation of PLA during the 10th Five-year plan period, No. 01Q138the Science and Technique Foundation of PLA during the 10th Five-year Plan period, No. 01MB135
文摘AIM: To investigate the transactivating effect of complete S protein of hepatitis B virus (HBV) and to construct a subtractive cDNA library of genes transactivated by complete S protein of HBV by suppression subtractive hybridization (SSH) technique and to clone genes associated with its transactivation activity, and to pave the way for elucidating the pathogenesis of hepatitis B virus infection. METHODS: pcDNA3.1(-)-complete S containing full-length HBV S gene was constructed by insertion of HBV complete S gene into BamH I/Kpn I sites. HepG2 cells were cotransfected with pcDNA3.1(-)-complete S and pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-galactosidase (β-gal). Suppression subtractive hybridization and bioinformatics techniques were used. The mRNA of HepG2 cells transfected with pcDNA3.Incomplete S and pcDNA3.1(-) empty vector was isolated, and detected for the expression of complete S protein by reverse transcription polymerase chain reaction (RT-PCR) method, and cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptors 1 and 2, respectively. After tester cDNA had been hybridized with driver cDNA twice and underwent nested PCR twice, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out within E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR) amplification. RESULTS: The complete S mRNA could be detected by RT-PCR in HepG2 cells transfected with the pcDNA3.1(-)-complete S. The activity of β-gal in HepG2 cells transfected with the pcDNA3.1(-)-complete s was 6.9 times higher than that of control plasmid. The subtractive library of genes transactivated by HBV complete S protein was constructed successfully. The amplified library contains 86 positive clones. Colony PCR showed that 86 clones contained DNA inserts of 200-1 000 bp, respectively. Sequence analysis was performed in 35 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 33 coding sequences were obtained. These cDNA sequences might be target genes transactivated by complete S protein of HBV. Moreover, two unknown genes were discovered, full length coding sequences were obtained by bioinformatics techniques, one of them was named complete S transactivated protein 1 (CSTP1) and registered in GenBank (AY553877). CONCLUSION: The complete S gene of HBV has a transactivating effect on SV40 early promoter. A subtractive cDNA library of genes transactivated by HBV complete S protein using SSH technique has been constructed successfully. The obtained sequences may be target genes transactivated by HBV complete S protein among which some genes coding proteins are involved in cell cycle regulation, metabolism, immunity, signal transduction, cell apoptosis and formation mechanism of hepatic carcinoma.
基金funded by Natural Science Foundation of Jian-gsu Province (BK2009660)Huaiyin Institute of Technology Foundation (35170706)
文摘[Objective] To obtain pure recombinant S1 and S2 of SARS S protein. [Method] Using asymmetric PCR and ligation with endonuclease, S1 and S2 fragments of SARSV HK strain S gene were synthesized. Then, these two fragments were inserted into plasmid pET28a to obtain recombinant vectors pET28a-S1 and pET28a-S2, respectively. These recombinant vectors were transformed into E. coli BL21, and expression of S1 and S2 fragments were induced by IPTG. The conditions of expression and purification were optimized. [Result] The S1 and S2 fragments were amplified and successfully expressed in E. coli. [Conclusion] This research provides detection antigens for follow-up development of SARS vaccine.
基金Supported by the National Natural Science Foundation of China, No. C03011402, No. C30070690the Science and Technique Foundation of PLA during the 9th Five-year plan period, No. 98D063 the Launching Foundation for Students Studying Abroad of PLA, No. 98H038 the Youth Science and Technique Foundation of PLA during the 10lh Five-year plan period, No. 01Q138and the Science & Technique Foundation of PLA during the 10th Five-year plan period, No. 01MB135
文摘AIM: To investigate the biological function of complete S protein and to look for proteins interacting with complete S protein in hepatocytes. METHODS: We constructed bait plasmid expressing complete S protein of HBV by cloning the gene of complete S protein into pGBKT7, then the recombinant plasmid DNA was transformed into yeast AH109 (a type). The transformed yeast AH109 was mated with yeast Y187 (α type) containing liver cDNA library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-α-gal for selection and screening. After extracting and sequencing of plasmids from positive (blue) colonies, we underwent sequence analysis by bioinformatics. RESULTS: Nineteen colonies were selected and sequenced. Among them, five colonies were Homo sapiens solute carrier family 25, member 23 (SLC25A23), one was Homo sapiens calrer.iculin, one was human serum albumin (ALB) gene, one was Homo sapiens metallothionein 2A, two were Homo sapiens betaine-homocysteine methyltransferase, three were Homo sapiens Na+ and H+coupled amino acid transport system N, one was Homo sapiens CD81 antigen (target of anti-proliferative antibody 1) (CD81), three were Homo sapiens diazepam binding inhibitor, two colonies were new genes with unknown function. CONCLUSION: The yeast-two hybrid system is an effective method for identifying hepatocyte proteins interacting with complete S protein of HBV. The complete S protein may bind to different proteins i.e., its multiple functions in vivo.
基金This research was funded by the National Natural Science Foundation of China(31900746 and 31802170)Postdoctoral Science Foundation(2019M660182).
文摘Porcine epidemic diarrhea(PED)caused by the porcine epidemic diarrhea virus(PEDV),is a severe infectious and devastating swine disease that leads to serious economic losses in the swine industry worldwide.An increased number of PED cases caused by variant PEDV have been reported in many countries since 2010.S protein is the main immunogenic protein containing some B-cell epitopes that can induce neutralizing antibodies of PEDV.In this study,the construction,expression and purification of Pseudomonas aeruginosa exotoxin A(PE)without domain Ⅲ(PE△Ⅲ)as a vector was performed for the delivery of PEDV S-A or S-B.PE(△Ⅲ)PEDV S-A and PE(△Ⅲ)PEDV S-B recombinant proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis.The immunogenicity of PEDV S-A and PEDV S-B subunit vaccines were evaluated in mice.The results showed that PEDV-S-B vaccine could not only induce specific humoral and Th1 type-dominant cellular immune responses,but also stimulate PEDV-specific mucosal immune responses in mice.PEDV-S-B subunit vaccine is a novel candidate mucosal vaccine against PEDV infection.
文摘C/EBP homologous protein, an important transcription factor during endoplasmic reticulum stress, participates in cell apoptosis mediated by endoplasmic reticulum stress. Previous studies have shown that C/EBP homologous protein mediates nerve injury during Alzheimer＇s disease, subarachnoid hemorrhage and spinal cord trauma. In this study, we introduced C/EBP homologous protein short hairpin RNA into the brains of ischemia/reperfusion rat models via injection of lentiviral vector through the left lateral ventricle. Silencing C/EBP homologous protein gene expression significantly reduced cerebral infarction volume, decreased water content and tumor necrosis factor-α and interleukin-1β mRNA expression in brain tissues following infarction, diminished the number of TUNEL-positive cells in the infarct region, decreased caspase-3 protein content and increased Bcl-2 protein content. These results suggest that silencing C/EBP homologous protein lessens cell apoptosis and inflammatory reactions, thereby protecting nerves.
文摘Meanwhile the outbreak of the Covid-19 since December, 2019 in China, it has killed more than a hundred thousand of people of all ages and sex across the globe in a short span of time. On the bases of this study the nearest family member of the virus and its receptor binding domain of S protein including its model structure and function of its active sites were naked through Multiple Sequence Alignment, modelling and molecular docking software accordingly its repository genome databases. The virus was genetically associated and molecular evolutionary related with (<em>RaTG</em>13) and it scores 96.12% homology with 99% query coverage followed by <em>bat-SL-CoVZC</em>45 and<em> bat-SL-CoVZXC</em>21 notch 89.12% and 88.65% respectively. However, SARS and MERS corona type virus those outbreak earlier respectively less likely family members of 2019-nCoV. Though the virus has a close genetic association with those previous SARS coronaviruses, and certainly the spike protein used as a binding receptor to fight against human receptor protein of ACE 2, but on the basis of FRODOC and HDOCK server analysis multi favorable active sites of S protein was discovered such GLN493 shown as a finest key in both model and possessed a unique traits on it resulting unexpected rate of transmission and number of people died while compared to the previous one. TYR500, ASN501, GLN498 and others residues preferably contemplate site also. In particular, the diversity of the virus in the world may be due to the genome structure of the virus and S gene changed over the time, across the world against to host of human genetic diversity, which may be more robust, and may be a new and unique feature. This is because it is characterized close to contact with distance divergence between wild type novel coronavirus which was risen from China against to the genomes from Lebanon, India, Italy, and USA and so on. Thus, the World Health Organization and its researchers should focus on immunologic research and effective drug and vaccine development that will help to address the epidemiology of the virus, which can provide a long-term solution.
