The paper was to obtain the VP1 protein of FMDV serotype A with high activity. With recombinant plasmid pMD19A-T-vp1 as the tem- plate, vpl gene fragment amplified by PCR was connected into prokaryotic expression vect...The paper was to obtain the VP1 protein of FMDV serotype A with high activity. With recombinant plasmid pMD19A-T-vp1 as the tem- plate, vpl gene fragment amplified by PCR was connected into prokaryotic expression vector pET28a to construct recombinant plasmid pET-A-vpl. The E. coli BL21 （DE3） strain containing recombinant plasmid pET-A-vpl were induced by IPTG. SDS-PAGE showed that VP1 protein was ex- pressed in the form of inclusion body, and its molecular weight was about 29 ku. Based on the optimizing IPTG concentration and expression time, the largest expression of VP1 protein was induced by 0.3 mmol/L IPTG for 6 h at 37 ℃. Western-Blot analysis indicated that the expression of VP1 protein could be specifically recognized by positive serum of FMDV serotype A. ELISA test showed that VP1 inclusion body protein had high activity after purification by washing and renaturation by urea concentration gradient dialysis.展开更多
A water-soluble polysaccharide, HM（41）, was obtained from Halenia elliptica D. Don by acidic ethanol fractionation and gel filtration. Its homogeneity was confirmed by chromatography using multiple systems. HM（41）...A water-soluble polysaccharide, HM（41）, was obtained from Halenia elliptica D. Don by acidic ethanol fractionation and gel filtration. Its homogeneity was confirmed by chromatography using multiple systems. HM（41） was composed of rhamnose（Rha）, arabinose（Ara）, xylose（Xyl）, mannose（Man）,galactose（Gal）, glucose（Glc） with a molar ratio of 1.0：5.5：1.8：3.0：9.4：21. The average molecular weight of HM（41） was approximately 1.17 ＊ 10~4. Periodate oxidation, Smith degradation, methylation and GC, IR,NMR, XRD, GC–MS analysis were used for the structural analysis of HM（41）. Its main chain was composed mainly of β-（1→4）Gal, β-（1→4）Glc and β-（1→6）Glc. β-（1 →4）Gal were substituted at 6-O and on average there were 14 branches among 23 main chain residues;（1→ 4）Glc had no branch;（1→6）Glc were substituted at 3-O and on average there were 9 branches among 14 main chain residues. The side chain was composed of（1→3,6）-Rha,（1→4）/（1→5）-Ara,（1 →4）/（1→5）-Xyl,（1→4,6）-Man and（1→2）-Glc. The terminal residue was composed of Ara, Xyl, Man, Gal, and Glc. Then, we demonstrated that HM and HM（41） had strong scavenging activities in vitro hydroxyl. Overall, HM and HM（41） may have potential applications in the antioxidants for medical and food industry.展开更多
Angiotensin-converting enzyme 2(ACE2) is not only an enzyme but also a functional receptor on cell membrane for severe acute respiratory syndrome coronavirus 2(SARS-CoV-2). Here, the activity of ACE2 in single living ...Angiotensin-converting enzyme 2(ACE2) is not only an enzyme but also a functional receptor on cell membrane for severe acute respiratory syndrome coronavirus 2(SARS-CoV-2). Here, the activity of ACE2 in single living cell is firstly determined using a nanokit coupled electrospray ionization mass spectrometry(nanokit-ESI-MS). Upon the insertion of a micro-capillary into the living h ACE2-CHO cell and the electrochemical sorting of the cytosol, the target ACE2 enzyme hydrolyses angiotensin II inside the capillary to generate angiotensin 1-7. After the electrospray of the mixture at the tip of the capillary, the product is differentiated from the substrate in molecular weight to achieve the detection of ACE2 activity in single cells. The further measurement illustrates that the inflammatory state of cells does not lead to the significant change of ACE2 catalytic activity, which elucidates the relationship between intracellular ACE2 activity and inflammation at single cell level. The established strategy will provide a specific analytical method for further studying the role of ACE2 in the process of virus infection, and extend the application of nanokit based single cell analysis.展开更多
基金Supported by NSFC-Joint Personnel Training Fund of Henan Province(U1204327)Special Fund for Construction of Provincial Key Laboratory in Henan Province(122300413217)
文摘The paper was to obtain the VP1 protein of FMDV serotype A with high activity. With recombinant plasmid pMD19A-T-vp1 as the tem- plate, vpl gene fragment amplified by PCR was connected into prokaryotic expression vector pET28a to construct recombinant plasmid pET-A-vpl. The E. coli BL21 （DE3） strain containing recombinant plasmid pET-A-vpl were induced by IPTG. SDS-PAGE showed that VP1 protein was ex- pressed in the form of inclusion body, and its molecular weight was about 29 ku. Based on the optimizing IPTG concentration and expression time, the largest expression of VP1 protein was induced by 0.3 mmol/L IPTG for 6 h at 37 ℃. Western-Blot analysis indicated that the expression of VP1 protein could be specifically recognized by positive serum of FMDV serotype A. ELISA test showed that VP1 inclusion body protein had high activity after purification by washing and renaturation by urea concentration gradient dialysis.
基金supported by the First-Class Discipline Construction Project for First-Class University of Minzu University of China (No. YLDX01013),111 Project (No. B08044)The ecological conservation technology integration and demonstration in north of Dzungaria (No. 2014BAC15B04)
文摘A water-soluble polysaccharide, HM（41）, was obtained from Halenia elliptica D. Don by acidic ethanol fractionation and gel filtration. Its homogeneity was confirmed by chromatography using multiple systems. HM（41） was composed of rhamnose（Rha）, arabinose（Ara）, xylose（Xyl）, mannose（Man）,galactose（Gal）, glucose（Glc） with a molar ratio of 1.0：5.5：1.8：3.0：9.4：21. The average molecular weight of HM（41） was approximately 1.17 ＊ 10~4. Periodate oxidation, Smith degradation, methylation and GC, IR,NMR, XRD, GC–MS analysis were used for the structural analysis of HM（41）. Its main chain was composed mainly of β-（1→4）Gal, β-（1→4）Glc and β-（1→6）Glc. β-（1 →4）Gal were substituted at 6-O and on average there were 14 branches among 23 main chain residues;（1→ 4）Glc had no branch;（1→6）Glc were substituted at 3-O and on average there were 9 branches among 14 main chain residues. The side chain was composed of（1→3,6）-Rha,（1→4）/（1→5）-Ara,（1 →4）/（1→5）-Xyl,（1→4,6）-Man and（1→2）-Glc. The terminal residue was composed of Ara, Xyl, Man, Gal, and Glc. Then, we demonstrated that HM and HM（41） had strong scavenging activities in vitro hydroxyl. Overall, HM and HM（41） may have potential applications in the antioxidants for medical and food industry.
基金supported by Ministry of Science and Technology of China(No.2017YFA0700500)the National Natural Science Foundation of China(Nos.22025403 and 21974060)+1 种基金Kuanren Talents Program of the Second Affiliated Hospital of Chongqing Medical UniversityYoung and Middle-aged Senior Medical Talents Studio of Chongqing。
文摘Angiotensin-converting enzyme 2(ACE2) is not only an enzyme but also a functional receptor on cell membrane for severe acute respiratory syndrome coronavirus 2(SARS-CoV-2). Here, the activity of ACE2 in single living cell is firstly determined using a nanokit coupled electrospray ionization mass spectrometry(nanokit-ESI-MS). Upon the insertion of a micro-capillary into the living h ACE2-CHO cell and the electrochemical sorting of the cytosol, the target ACE2 enzyme hydrolyses angiotensin II inside the capillary to generate angiotensin 1-7. After the electrospray of the mixture at the tip of the capillary, the product is differentiated from the substrate in molecular weight to achieve the detection of ACE2 activity in single cells. The further measurement illustrates that the inflammatory state of cells does not lead to the significant change of ACE2 catalytic activity, which elucidates the relationship between intracellular ACE2 activity and inflammation at single cell level. The established strategy will provide a specific analytical method for further studying the role of ACE2 in the process of virus infection, and extend the application of nanokit based single cell analysis.
