The VP1 protein of foot-and-mouth disease virus serotype A was prokaryotically expressed and purified to replace the traditional virus antigen for estab- lishing a fast, safe, effective indirect ELISA method, so as to...The VP1 protein of foot-and-mouth disease virus serotype A was prokaryotically expressed and purified to replace the traditional virus antigen for estab- lishing a fast, safe, effective indirect ELISA method, so as to detecting antibody of foot-and-mouth disease virus serotype A. Western-Blot test showed that the VP1 recombinant protein could be used as detective antigen as it can be specifically recognized by bovine positive serum of FMDV serotype A. By employing matrix titra- tion method, the optimal parameters were obtained as follows: 1 mg/L VP1 protein as coating antigen, Vserum:Vblocking solution = 1:50 dilution for serum and Vsecondary enzyme-linked antibedies:Vblocking solution ---1:2 000 for enzyme combined antibodies. The results showod that the sensitivity and specificity of this method were 94.32% and 99.09% respectively, the coefficients of variations in intra-assay and inter-assay reproducibility tests was lower than 8%. Compared with liquid phase blocking ELISA kits, the agreement of 201 serum samples reached 92.54%. The VP1-ELISA method established here is specific, sensitive, stable and simple, which can be used to monitor the antibody level of FMD serotype A.展开更多
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
Variable Heavy-chain Homodimer (VHH) or Nanobody is a recombinant dromedary antibody fragment which is classified as the smallest antibody fragments with the highest binding affinity and specificity of the original wh...Variable Heavy-chain Homodimer (VHH) or Nanobody is a recombinant dromedary antibody fragment which is classified as the smallest antibody fragments with the highest binding affinity and specificity of the original whole antibody. In this study the Expression of Nanobodies in <em>E. coli</em> WK6 cell periplasm was performed. The protein expression and purity was and analyzed by Affinity Chromatography, SDS PAGE and Western Blot. Upon elution with Imidazole, the concentrations observed using the OD<sub>280</sub> nm of the eluted fractions EI, E2 and E3 were observed to be 0.42 μg/ml, 0.13 μg/ml and <span style="white-space:nowrap;">−</span>0.46 μg/ml respectively. This gives an Antilog of 7.88 kDa which showed the calculated molecular size of our band. The SDS-PAGE gel reading was confirmed using Western blot analysis and illustrated as the specific binding of the mouse Anti-His antibody to the Histidine tag of the Nanobody. The Nanobody protein expression was then analyzed further with western blotting showed a strong signal at the region corresponding to the 15 kDa marker indicating presence of the Nanobody gene. This was taken as further confirmation of the protein expression from the bacterial cells.展开更多
Fertilization in mammals requires the successful completion of a sequence of steps, starting with the transport of gametes in the reproductive tract and ending with sperm-egg membrane fusion to produce a zygote. Altho...Fertilization in mammals requires the successful completion of a sequence of steps, starting with the transport of gametes in the reproductive tract and ending with sperm-egg membrane fusion to produce a zygote. Although some integrin subunits are known to be associated with the plasma membrane of some mammalian oocytes and spermatozoa, the presence of α6 integrin on bovine oocytes with intact zona pellucida has not been reported. The present study was undertaken to evaluate the expression of α6 integrin subunit in bovine oocyte and to determine if in vitro binding to the zona pellucida and fertilization were affected by treating oocytes with α6 integrin subunit antibody. The α6 integrin subunit was identified on the bovine oocyte by immunocytochemistry. In vitro fertilization was significantly decreased when in vitro matured bovine oocytes were pre-incubated with α6 integrin subunit antibody at concentration 5 and 20 μg/mL, and spermoocyte binding increased. These studies demonstrated the presence of α6 integrin subunit on bovine oocyte, and its importance in fertilization and polyspermy.展开更多
Two hundred and forty specific pathogen free leghorn chickens were randomly divided into four groups and reared in isolated pens. The tested chickens were negative to infectious bursal disease virus (IBDV) at 25 d o...Two hundred and forty specific pathogen free leghorn chickens were randomly divided into four groups and reared in isolated pens. The tested chickens were negative to infectious bursal disease virus (IBDV) at 25 d old. Group 1 was treated with saline, whereas Groups 2, 3, and 4 were inoculated with 0.3 mL IBDV suspension intranasally the next day. Groups 3 and 4 were also administered with Astragalus polysaccharides (APS) intramuscularly twice daily at 5 or 10 mg kg-1 BW, respectively, until 31 d old. The erythrocyte-C3b receptor rosette rate (E-C3bRR) and the erythrocyte-C3b immune complex rosette rate (E-ICRR) were measured at 25, 29, 32, 35, and 38 d old. The results showed that IBDV significantly reduced E-C3bRR and E-ICRR when compared with the control group (P 〈 0.05), while simultaneous administration of APS with 1BDV maintained E-C3bRR at similar levels to the control group (P 〉 0.05) and increased E-ICRR when compared with the control group and the group non-treated with APS (P 〈 0.05). APS treatment reduced the morbidity and mortality of chickens inoculated with IBDV (P 〈 0.05). The results suggest that APS may enhance the immune adherence of chickens erythrocytes by affecting the activity and/or the number of complement receptors on the erythrocyte membrane. These findings can be beneficial in providing an understanding of the basic mechanisms required for the rational application of APS in modern medicine.展开更多
Porcine reproductive and respiratory syndrome(PRRS) is considered to be one of the most important infectious diseases impacting the swine industry and is characterized by reproductive failure in late term gestation in...Porcine reproductive and respiratory syndrome(PRRS) is considered to be one of the most important infectious diseases impacting the swine industry and is characterized by reproductive failure in late term gestation in sows and respiratory disease in pigs of all ages.The nonstructural protein 9 gene,Nsp9,encoding the RNA-dependent RNA polymerase,is generally regarded as fairly conserved when compared to other viral proteins.Antibodies against Nsp9 will be of great importance for the diagnosis and treatment of the causal agent,PRRS virus.A study was undertaken to generate polyclonal antibodies against the immunodominant Nsp9.For this purpose,the Nsp9 was expressed in Escherichia coli and subsequently used as an antigen to immunize New Zealand rabbits.Antiserum was identified via an indirect ELISA,and then verified based on the ability to react with both naturally and artificially expressed Nsp9.Results of virus neutralization test showed that this antiserum could not neutralize the PRRSV.Nevertheless,this antiserum as a diagnostic core reagent should prove invaluable for further investigations into the mechanism of PRRS pathogenesis.展开更多
Supraventricular tachyarrhythmias may be caused by macroreentry circuits involving the AV node and accessory pathways. This paper reports a case of suspected orthodromic atrioventricular reciprocating tachycardia in a...Supraventricular tachyarrhythmias may be caused by macroreentry circuits involving the AV node and accessory pathways. This paper reports a case of suspected orthodromic atrioventricular reciprocating tachycardia in an 18-month-old Dalmatian admitted with dyspnea and a lifelong history of fatigue. Cardiac auscultation documented a regular fast pace with no heart murmurs. The electrocardiogram characteristics were consistent with supraventricular tachycardia, with very regular RR interval and narrow QRS complexes. At lead II, we identified negative P waves buried within the ST segment, which resulted in a RP-to-PR ratio of 0.60, but in aVR these P waves were positive, suggesting a retrograde conduction of electrical impulses throughout the atrial myocardium. The echocardiographic study showed volume overload, and a decreased fractional shortening was calculated when SVT was sustained, highlighting its impact on systolic function. This is likely the first description of an orthodromic atrioventricular reciprocating tachycardia in a Dalmatian, and although cardiac mapping was not available to confirm this suspicion, all electrocardiograpic features were supportive of such arrhythmia.展开更多
Whenever there is no adequate DNA replication <em>in vitro</em>, there are alternatives strategies to insert a piece of DNA into a convenient replicon such as small plasmids and bacteriophages. These are c...Whenever there is no adequate DNA replication <em>in vitro</em>, there are alternatives strategies to insert a piece of DNA into a convenient replicon such as small plasmids and bacteriophages. These are called vectors or cloning vehicles. These days, there is a high demand to manufacture recombinant and nanobodies which are required in biomedical research and for therapeutic and diagnostic purposes. In order to do so, <em>E. coli</em>, insect cells, or mammalian cells have been used to express and purify protein for nanobody production. This paper explains the experimental trials on the cloning of the Nanobody cDNA mock-up in pHEN6c plasmid vector from the subcultured <em>E. coli</em> sample taken from the lab. The concentration and purity of DNA plasmid were evaluated by UV spectrophotometer and agarose gel electrophoresis following plasmid DNA Purification from <em>E. coli</em> by alkaline lysis. Based on this, the concentration of the isolated pHEN6c plasmid DNA was found 44.7 ng/μl or 0.0447 μg/μl. Whereas, the purity at absorbance (A260/A280) was 0.893/0.501 = 1.78. Moreover, its yield was 2.235 μg. In addition, its transformation efficiency was 21.68 μg/μl. On the other hand, the molecular weight of the Nanobody and vector were 569 and 2717 respectively. Generally, most of the protocols used to clone a fragment of DNA, might not work very well with PCR products.展开更多
基金Supported by Joint Funds of the NSFC and Henan Province(U1204327)Henan Provincial Key Laboratory Construction(122300413217)
文摘The VP1 protein of foot-and-mouth disease virus serotype A was prokaryotically expressed and purified to replace the traditional virus antigen for estab- lishing a fast, safe, effective indirect ELISA method, so as to detecting antibody of foot-and-mouth disease virus serotype A. Western-Blot test showed that the VP1 recombinant protein could be used as detective antigen as it can be specifically recognized by bovine positive serum of FMDV serotype A. By employing matrix titra- tion method, the optimal parameters were obtained as follows: 1 mg/L VP1 protein as coating antigen, Vserum:Vblocking solution = 1:50 dilution for serum and Vsecondary enzyme-linked antibedies:Vblocking solution ---1:2 000 for enzyme combined antibodies. The results showod that the sensitivity and specificity of this method were 94.32% and 99.09% respectively, the coefficients of variations in intra-assay and inter-assay reproducibility tests was lower than 8%. Compared with liquid phase blocking ELISA kits, the agreement of 201 serum samples reached 92.54%. The VP1-ELISA method established here is specific, sensitive, stable and simple, which can be used to monitor the antibody level of FMD serotype A.
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.
文摘Variable Heavy-chain Homodimer (VHH) or Nanobody is a recombinant dromedary antibody fragment which is classified as the smallest antibody fragments with the highest binding affinity and specificity of the original whole antibody. In this study the Expression of Nanobodies in <em>E. coli</em> WK6 cell periplasm was performed. The protein expression and purity was and analyzed by Affinity Chromatography, SDS PAGE and Western Blot. Upon elution with Imidazole, the concentrations observed using the OD<sub>280</sub> nm of the eluted fractions EI, E2 and E3 were observed to be 0.42 μg/ml, 0.13 μg/ml and <span style="white-space:nowrap;">−</span>0.46 μg/ml respectively. This gives an Antilog of 7.88 kDa which showed the calculated molecular size of our band. The SDS-PAGE gel reading was confirmed using Western blot analysis and illustrated as the specific binding of the mouse Anti-His antibody to the Histidine tag of the Nanobody. The Nanobody protein expression was then analyzed further with western blotting showed a strong signal at the region corresponding to the 15 kDa marker indicating presence of the Nanobody gene. This was taken as further confirmation of the protein expression from the bacterial cells.
文摘Fertilization in mammals requires the successful completion of a sequence of steps, starting with the transport of gametes in the reproductive tract and ending with sperm-egg membrane fusion to produce a zygote. Although some integrin subunits are known to be associated with the plasma membrane of some mammalian oocytes and spermatozoa, the presence of α6 integrin on bovine oocytes with intact zona pellucida has not been reported. The present study was undertaken to evaluate the expression of α6 integrin subunit in bovine oocyte and to determine if in vitro binding to the zona pellucida and fertilization were affected by treating oocytes with α6 integrin subunit antibody. The α6 integrin subunit was identified on the bovine oocyte by immunocytochemistry. In vitro fertilization was significantly decreased when in vitro matured bovine oocytes were pre-incubated with α6 integrin subunit antibody at concentration 5 and 20 μg/mL, and spermoocyte binding increased. These studies demonstrated the presence of α6 integrin subunit on bovine oocyte, and its importance in fertilization and polyspermy.
文摘Two hundred and forty specific pathogen free leghorn chickens were randomly divided into four groups and reared in isolated pens. The tested chickens were negative to infectious bursal disease virus (IBDV) at 25 d old. Group 1 was treated with saline, whereas Groups 2, 3, and 4 were inoculated with 0.3 mL IBDV suspension intranasally the next day. Groups 3 and 4 were also administered with Astragalus polysaccharides (APS) intramuscularly twice daily at 5 or 10 mg kg-1 BW, respectively, until 31 d old. The erythrocyte-C3b receptor rosette rate (E-C3bRR) and the erythrocyte-C3b immune complex rosette rate (E-ICRR) were measured at 25, 29, 32, 35, and 38 d old. The results showed that IBDV significantly reduced E-C3bRR and E-ICRR when compared with the control group (P 〈 0.05), while simultaneous administration of APS with 1BDV maintained E-C3bRR at similar levels to the control group (P 〉 0.05) and increased E-ICRR when compared with the control group and the group non-treated with APS (P 〈 0.05). APS treatment reduced the morbidity and mortality of chickens inoculated with IBDV (P 〈 0.05). The results suggest that APS may enhance the immune adherence of chickens erythrocytes by affecting the activity and/or the number of complement receptors on the erythrocyte membrane. These findings can be beneficial in providing an understanding of the basic mechanisms required for the rational application of APS in modern medicine.
基金supported by the National Natural Science Foundation of China(31272564)the Modern Agricultural Industry Technology System(CARS-36)Special Fund for Agro-scientific Research in the Public Interest(201203039)
文摘Porcine reproductive and respiratory syndrome(PRRS) is considered to be one of the most important infectious diseases impacting the swine industry and is characterized by reproductive failure in late term gestation in sows and respiratory disease in pigs of all ages.The nonstructural protein 9 gene,Nsp9,encoding the RNA-dependent RNA polymerase,is generally regarded as fairly conserved when compared to other viral proteins.Antibodies against Nsp9 will be of great importance for the diagnosis and treatment of the causal agent,PRRS virus.A study was undertaken to generate polyclonal antibodies against the immunodominant Nsp9.For this purpose,the Nsp9 was expressed in Escherichia coli and subsequently used as an antigen to immunize New Zealand rabbits.Antiserum was identified via an indirect ELISA,and then verified based on the ability to react with both naturally and artificially expressed Nsp9.Results of virus neutralization test showed that this antiserum could not neutralize the PRRSV.Nevertheless,this antiserum as a diagnostic core reagent should prove invaluable for further investigations into the mechanism of PRRS pathogenesis.
文摘Supraventricular tachyarrhythmias may be caused by macroreentry circuits involving the AV node and accessory pathways. This paper reports a case of suspected orthodromic atrioventricular reciprocating tachycardia in an 18-month-old Dalmatian admitted with dyspnea and a lifelong history of fatigue. Cardiac auscultation documented a regular fast pace with no heart murmurs. The electrocardiogram characteristics were consistent with supraventricular tachycardia, with very regular RR interval and narrow QRS complexes. At lead II, we identified negative P waves buried within the ST segment, which resulted in a RP-to-PR ratio of 0.60, but in aVR these P waves were positive, suggesting a retrograde conduction of electrical impulses throughout the atrial myocardium. The echocardiographic study showed volume overload, and a decreased fractional shortening was calculated when SVT was sustained, highlighting its impact on systolic function. This is likely the first description of an orthodromic atrioventricular reciprocating tachycardia in a Dalmatian, and although cardiac mapping was not available to confirm this suspicion, all electrocardiograpic features were supportive of such arrhythmia.
文摘Whenever there is no adequate DNA replication <em>in vitro</em>, there are alternatives strategies to insert a piece of DNA into a convenient replicon such as small plasmids and bacteriophages. These are called vectors or cloning vehicles. These days, there is a high demand to manufacture recombinant and nanobodies which are required in biomedical research and for therapeutic and diagnostic purposes. In order to do so, <em>E. coli</em>, insect cells, or mammalian cells have been used to express and purify protein for nanobody production. This paper explains the experimental trials on the cloning of the Nanobody cDNA mock-up in pHEN6c plasmid vector from the subcultured <em>E. coli</em> sample taken from the lab. The concentration and purity of DNA plasmid were evaluated by UV spectrophotometer and agarose gel electrophoresis following plasmid DNA Purification from <em>E. coli</em> by alkaline lysis. Based on this, the concentration of the isolated pHEN6c plasmid DNA was found 44.7 ng/μl or 0.0447 μg/μl. Whereas, the purity at absorbance (A260/A280) was 0.893/0.501 = 1.78. Moreover, its yield was 2.235 μg. In addition, its transformation efficiency was 21.68 μg/μl. On the other hand, the molecular weight of the Nanobody and vector were 569 and 2717 respectively. Generally, most of the protocols used to clone a fragment of DNA, might not work very well with PCR products.