ABSTRACT
Objective:In order to establish ELISA method for testing tobacco mosaic virus (TMV) infecting Zingiber officinale Rosc.Methods: Tobacco mosaic virus(TMV) infecting Zingiber officinale Rosc was tested by RT-PCR.Zingiber officinale Rosc leaves which only contained TMV were choiced.TMV particle was purified by centrifugation method.TMV CP was purified through preparation electrophoresis including 12％ SDS-PAGE first and then 5％-20％ gradient SDS-PAGE.Polyacrylamide gel contained TMV CP was ground into suspension.Mice were immuned with the suspension and antiserum was obtained.Antiserum quality was tested by Western blot and ELISA test.IgG was purified through affinity chromatography method.IgG solution was concentrated and dialyzed to a suitable concentration.The IgG then mixed with glycerol.Results: IgG in antiserum only combined with TMV CP protein and it could combine with nature TMV particle CP protein.Its quality was up to standard.Conclusion: Establishment of ELISA method for testing TMV infecting Zingiber offcinale Rosc is successfull by using this IgG.
ABSTRACT
3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) is the key enzyme in L-serine biosynthesis and its coding gene is serA. PGDH is feedback inhibited by L-serine. In order to relieve the feedback-inhibition of PGDH by L-serine, H344 or D346 or D364 were chosen for site directed mutagenesis. The mutants were generated by the standard QuikChange mutagenesis, further subcloned into expression vector pT7-7 and transformed into Escherichia coli BL21 (DE3) cells. The recombinant cells were collected after cultured in LB media post induced by isopropyl beta-Dthiogalactopyranoside. The enzymes were purified by anion exchange chromatography, and SDS-PAGE showed that the purified enzymes were homogenous. Enzyme characterization indicated that the mutant enzyme showed similar activity, optimal temperature, and optimal pH as that of the wild-type enzyme. Moreover, feedback inhibition study showed that the activity of the double mutant (N346A/H344A) could remain 96% in the presence of serine up to 160 mmol/L, whereas the activity of the wild-type enzyme remains only 50% in the presents of serine of 7 μmol/L, thus successfully relieving the feedback inhibition of PGDH with its activity remained.
Subject(s)
Electrophoresis, Polyacrylamide Gel , Escherichia coli , Escherichia coli Proteins , Genetics , Industrial Microbiology , Mutagenesis, Site-Directed , Phosphoglycerate Dehydrogenase , Genetics , SerineABSTRACT
Objective:In order to look for a good method for preparation of hemorrhagin antiserum. Methods: Three kinds of hemorrhagins including AaH Ⅰ, AaH Ⅱ, and AaH Ⅳ were purified from Agkistrodon acutus venom according to predecessors's methods and crude AaH Ⅰ, AaH Ⅱ and AaH Ⅳ were obtained. Preparation electrophoresis was used to purify AaH Ⅰ,AaH Ⅱand AaH Ⅳ further. As for an hemorrhagin, six different dyeing methods were used to dye PAGE gel and the gel contained hemorrhagin was obtained respectively. The ground gel contained hemorrhagin was used to immune mice and its antiserum was obtained. Antiserums quality was tested through ELISA test and neutralization of the hemorrhagic activities of corresponding hemorrhagin. Results:Effective IgG concentration in different antiserum was different and effective IgG made through non toxic type protein fast stain reagent kit was higher than others. Conclusion:Non toxic type protein fast stain reagent kit is the best dyeing method among the six dyeing methods.
ABSTRACT
To study the long-term toxicity of arginine esterase from Agkistrodon halys ussuriensis venom for the clinical application of arginine esterase in the future. Beagle dogs were used as experimental animals and were divided into control group, arginine esterase low dose group (0.06 u/kg), the middle dose group (0.18 u/kg) and high dose group (0.36 u/kg). Every group consisted of four dogs. The arginine esterase was given intravenously once a day for 180 days. Then three dogs in each group were sacrificed and the fourth one was fed without injecting arginine esterase for 15 days. The toxic reactions during treatment and recovery period were determined by evaluating and comparing the general criteria ( including locomotor activity, growth rate, appetite and death rate), clinical criteria (including blood test and urine test), pathological dissection and viscera coefficient of the treated animals and the control animals. There were no significant differences in general criteria. The clinical criteria of the treated animals were the same as those of the control animals except liver function. There were no significant differences in pathological dissection and viscera coefficient between the treated animals and the control animals except livers. The livers in high dose arginine esterase treated animals were swollen and vacuolated and there was significant difference in liver coefficient between them (P<0.05). The toxic symptom of liver disappeared after withdrawal of treatment. From these results, the non-toxic dose of arginine esterase for dogs was estimated to be 0.18 u/kg under the present study conditions and is about 15 times the clinical dosage for using the drug "Qingshuanmei" of which the main component is arginine esterase. The long-term toxicity test result indicates that the toxicity of pure arginine esterase is lower than that of "Qingshuanmei", suggesting that clinical use of the arginine esterase is safe.