Development and verification of a sulfosalicylic acid spectrophotometric method for determination of iron ion in medium for producing diphtheria toxin / 中国生物制品学杂志

@#Objective To develop and verify a sulfosalicylic acid spectrophotometric method for the determination of trace iron ions in diphtheria toxin medium,and apply it preliminarily.Methods The maximum absorbance of the complex of iron and sulfosalicylic acid was scanned by full spectrum;A method for the determination of iron ion in culture medium was developed by linear regression between the absorbance of the complex and the content of iron ion,and the stability,accuracy and precision of the method were verified.The effects of Ca~(2+),Mg~(2+),K~+,Na~+ and reactants on the method were investigated.Spectrophotometric method with sulfosalicylic acid was used to determine trace iron in the self-made and commercial medium for diphtheria toxin production.Sulfosalicylic acid spectrophotometry,ferrizine colorimetry and o-phenanthroline spectrophotometry were used to detect iron content in two kinds of culture media(beef trypsin digestion liquid and 5% polypeptone),and the detection results of the three methods were compared.Results The complex of iron and sulfosalicylic acid showed the maximum absorbance at the wavelength of 425 nm;There was a good linear relationship between the absorbance and concentration of iron ion in the range of 1～0.05 μg/mL,the detection limit was 0.05 μg/mL,and the standard equation was:Y=0.027 9 X+0.046 1,R~2> 0.99;The coefficients of variation(CVs) of A_(425) value of each concentration of standards measured every 5 min were less than 5%;Low(0.05 μg/mL),medium(0.5 μg/mL)and high concentration(1 μg/mL) of Fe~(3+) standard solutions were continuously determined for 3 times.The CVs of 9groups of each concentration measured in parallel were all less than 5% and the recovery rates were higher than 95%;Ca~(2+),Mg~(2+),K~+,Na~+,15 μL of sulfo salicylic acid(20%) and 50 μL of ammonia hydroxide(1:1) showed no interference in the method;The results of toxin-producing medium were consistent with those of diphtheria bacteria;The results of the three detection methods were consistent.Conclusion The developed spectrophotometric method with sulfo salicylic acid can determine the content of trace iron ions in diphtheria toxin medium accurately and effectively.

Prokaryotic expression and one-step purification of soluble diphtheria toxin variant CRM197 / 基础医学与临床

Objective To construct a novel prokaryotic expression system, in which cross-reacting material 197 (CRM197) can be expressed in a soluble form in Escherichia coli(E. coli) cytoplasm and purified simply by one-step Ni-NTA affinity purification. Methods The CRM197 coding sequence was cloned into the prokaryotic expres-sion vector pET-32a(+) as an fusion protein with Trx tag,the HRV3C(human rhinovirus 3C) protease recognition sequence and 6 histidine sequence were added to the N-terminal of CRM197.HRV3C protease gene was cloned into another prokaryotic expression plasmid pGArasd. Both plasmids were co-transformed into E. coli Origami B (DE3) and induced mildly at 15℃. CRM197 recombinant protein was purified by Ni-NTA affinity matrix. Results The free soluble His-tagged CRM197 protein was released by cleavage of the accompanying expressed HRV3C protease after the CRM197 fusion protein was expressed. After one-step affinity purification recombinant CRM197 protein with a purity of almost 95% was obtained. Outcoming of the final preparation incubated with DNA indicated the pu-rified CRM197 recombinant protein has deoxyribonuclease activity. Conclusions By constructing a novel double-plasmid auto-cleavage prokaryotic expression system in this study, the production process of obtaining soluble CRM197 recombinant protein in E. coli has been simplified,with expression and purification efficiency improved and the production cost reduced.

Expression, purification and characterization of diphtheria toxin mutant CRM197 in Eschrichia coli / 生物工程学报

CRM197 (cross-reacting material 197), a non-toxic mutant of diphtheria toxin, has wide application potential in biopharmaceuticals. However, it is difficult to express CRM197 in bacteria other than Corynebacterium diphtheriae. Here we proposed a new alternative method to produce soluble CRM197 without label in Escherichia coli. In particular, a synthetic gene coding for CRM197, optimized for E. coli codon usage, was cloned in the pET32a (+) vector. Accordingly, the over-expression of the protein was simply induced with IPTG in E. coli BL21 (DE3). The target protein was soluble and accounted for about 40% of the total protein in the supernatant. Following an ultrasonic cytolysis step, the recombinant protein was purified by anion exchange, affinity and desalting chromatography and the purity of the final preparation reached 95%. Cytotoxicity tests showed that the IC₅₀ value of CRM197 was 2.1×10⁷ times the IC₅₀ value of diphtheria toxin, and 9.6 times the IC50 value of diphtheria toxoid, telling that the target protein is safe and non-toxic. Subsequently, we found that both the high dose (20 μg) and the low dose (2 μg) of CRM197 were equally efficient in inducing an immune response against diphtheria toxiod in mice, and the antibodies titer of mice after three immunizations with low dose could reach 1:409 600. In conclusion, our findings provide a highly efficient strategy for the rapid production and purification of unlabeled and soluble recombinant CRM197 in E. coli, with good immunogenicity and safety.

Effects of Different Doses of Diphtheria Toxin on the Cochlear Structure and AuditoryFunction in Wildtype Auditory-Mature Mice / 听力学及言语疾病杂志

Objective To investigate the effects of different doses of diphtheria toxin on cochlear structure and auditory function of adult wildtype mice.Methods The auditory-mature wild type C57BL/6J mice 4 weeks old were randomly devided into 50 ng/g group, 100 ng/g group and control group.C57BL/6J mice in the 50 ng/g or 100 ng/g group were injected 50 ng/g or 100 ng/g diphtheria toxin intraperitoneally for one time, respectively, and the control mice were injected equal volume of normal saline for one time.Then we investigated the ABR threshold change and morphological change of inner and outer hair cell and spiral ganglion neuron 7 days after the injection.Results At 7 day post diphtheria toxin injection compared with those of in control group, in the 50 ng/g group, there was no threshold elevation across frequencies(8 kHz ABR threshold was 20.0±3.78 and 20.83±2.04 dB SPL for 50 ng/g and control respectively), and no loss of inner and outer hair cells (for both groups, the HC loss rates were 0.3%~1%) or SGN (the SGN density was 39.45±3.65, 41.03±3.73/105 μm2, in 50 ng/g and control, respectively).However, the 100 ng/g group, compared with those of in control group, the ABR threshold (8 kHz ABR threshold was 63.0±4.47 dB SPL, respectively)was significantly elevated across each frequency(t=19.62,P<0.001), and there was significant loss of outer hair cell (the loss rate of IHC and OHC was 0.5%±0.1%, 10.7%±0.3%, respectively), which was 10% loss in the apical, middle and basal turn(t=42.219,P<0.001).And the loss of spiral ganglion neuron (the SGN density was 25.55±3.66/105 μm2) was 38%, which was significantly different from the control(t=10.985,P<0.001).Conclusion High dose injection of diphtheria toxin can cause loss of outer hair cell and spiral ganglion neuron in wild type auditory-mature C57BL/6J mice.

Persistence of Corynebacterium diphtheriae in Delhi & National Capital Region (NCR).

Despite the introduction of mass immunization, diphtheria continues to play a major role as a potentially lethal infectious disease in many countries. Delay in the specific therapy of diphtheria may result in death and, therefore, accurate diagnosis of diphtheria is imperative. This study was carried out at National Centre for Disease Control (NCDC), Delhi, India, on samples of suspected diphtheria cases referred from various government hospitals of Delhi and neighbouring areas during 2012-2014. Primary identification of Corynebacterium diphtheriae was done by standard culture, staining and biochemical tests followed by toxigenicity testing by Elek’s test on samples positive for C. diphtheriae. The results showed persistence of toxigenic C. diphtheriae in our community indicating the possibility of inadequate immunization coverage.

Multiplex polymerase chain reaction to identify and determine the toxigenicity of Corynebacterium spp with zoonotic potential and an overview of human and animal infections

Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis constitute a group of potentially toxigenic microorganisms that are related to different infectious processes in animal and human hosts. Currently, there is a lack of information on the prevalence of disease caused by these pathogens, which is partially due to a reduction in the frequency of routine laboratory testing. In this study, a multiplex polymerase chain reaction (mPCR) assay that can simultaneously identify and determine the toxigenicity of these corynebacterial species with zoonotic potential was developed. This assay uses five primer pairs targeting the following genes: rpoB (Corynebacterium spp), 16S rRNA (C. ulcerans and C. pseudotuberculosis), pld (C. pseudotuberculosis), dtxR (C. diphtheriae) and tox [diphtheria toxin (DT) ]. In addition to describing this assay, we review the literature regarding the diseases caused by these pathogens. Of the 213 coryneform strains tested, the mPCR results for all toxigenic and non-toxigenic strains of C . diphtheriae, C. ulcerans and C. pseudotuberculosis were in 100% agreement with the results of standard biochemical tests and PCR-DT. As an alternative to conventional methods, due to its advantages of specificity and speed, the mPCR assay used in this study may successfully be applied for the diagnosis of human and/or animal diseases caused by potentially toxigenic corynebacterial species.

Diphtheria Antibodies and T lymphocyte Counts in Patients Infected with HIV-1

We assessed the IgG levels anti-diphtheria (D-Ab) and T cell counts (CD4+ and CD8+) in HIV-1 infected subjects undergoing or not highly active antiretroviral therapy (HAART). Approximately 70% of all HIV-1 patients were unprotected against diphtheria. There were no differences in D-Ab according to CD4 counts. Untreated patients had higher D-Ab (geometric mean of 0.62 IU/ml) than HAART-patients (geometric mean of 0.39 IU/ml). The data indicated the necessity of keeping all HIV-1 patients up-to-date with their vaccination.

Dimensionamiento de los sistemas de microfiltración y ultrafiltración para la producción de la Toxina Diftérica elaborada en el Instituto Nacional de Higiene “Rafael Rangel” / Microfiltration and ultrafiltration sistems dimension for using in Difteria Toxin production in the Instituto Nacional de Higiene "Rafael Rangel"

Se evaluó el uso de la tecnología de Flujo de Filtración Tangencial (FFT), para obtener la toxina diftérica a partir de cultivos de la bacteria Corynebacterium diphtheriae, usando el proceso de Microfiltración (MF), para eliminar el paquete celular y posteriormente, a partir del filtrado obtenido, concentrar y diafiltrar la toxina diftérica usando el proceso de Ultrafiltración (UF). Se determinaron características de los filtros, condiciones de trabajo y dimensionamiento de los equipos a adquirir para la producción industrial de Toxina Diftérica. Se evaluaron el flujo, tiempo, rendimiento del proceso y las características del producto obtenido, utilizando cultivos con Toxina Diftérica en un equipo de filtración de laboratorio, diseñado para producir el efecto de FFT. Seseleccionó las membranas tipo cassettes, formato Médium Screen, porosidad 0,2 μm, como las adecuadas para el proceso de MF, ya que mostraron 100% de transmisión de la Toxina Diftérica, ausencia de restos celulares y flujo promedio de filtrado de 9.16 L/m2h. Así mismo, se seleccionaron las membranas tipo cassettes, formato Omega, porosidad 10 y 30 kDa, como las adecuadas para el proceso de UF, ya que mostraron 100% de recuperación de la toxina, ausencia de toxina en el filtrado y adecuados flujos de filtrado (97,5 y 125,9 L/m2h, respectivamente), Estos resultaron permitieron dimensionar, considerando las variables a utilizar en la producción industrial (Volumen 650 a 950 Litros, Tiempo de Procesos, 3 a 5 horas), el área de filtración de los equipos de MF y UF a adquirir, estimados en 20m2 y 5m2, respectivamente.

Tangential Flow Filtration (TFF) technology was evaluated to process diphtheria toxin which is produced by Cory ne - bacterium diphtheriae bacterium. Microfiltration (MF) is used to retain cells while allowing passage of the toxin to the filtrate stream. The filtrate is collected and further pro - cessed by Ultrafiltration (UF) to concentrate the toxin and to maximize the wash of small species by a Diafiltration step. Both, MF and UF processes were evaluated to specify the filters and corresponding critical process parameters to scale-up the application. As part of the evaluation, flow rate, processing time, yield and product attributes were characterized. The cell harvest containing the diphtheria toxin was processed using a laboratory scale TFF system designed to product the TFF effect. The evaluation demonstrated that a cassette in medium screen format and membrane with 0.2 μm pore is the right selection for the MF step. It showed 100% of toxin transmission without the presence of cellular debris and average process flux of 9.16 L/m2h. The UF step was conducted using the same laboratory equipment with cassettes in medium screen format with pores of 10 and 30 kD. It showed 100% retention of the toxin with a process flux of 97,5 and 125,9 L/m2h, respectively. These results were used to scale-up the application to process the industrial volume of 650 a 950 liters between 3 to 5 hours of processing time. Membrane area sizing of MF and UF to be acquired is estimated in 20 m2 and 5m2, respectively.

Expression and purification of the immunogenically active fragment B of the Park Williams 8 Corynebacterium diphtheriae strain toxin

The construction of a hexahistidine-tagged version of the B fragment of diphtheria toxin (DTB) represents an important step in the study of the biological properties of DTB because it will permit the production of pure recombinant DTB (rDTB) in less time and with higher yields than currently available. In the present study, the genomic DNA of the Corynebacterium diphtheriae Park Williams 8 (PW8) vaccine strain was used as a template for PCR amplification of the dtb gene. After amplification, the dtb gene was cloned and expressed in competent Escherichia coli M15™ cells using the expression vector pQE-30™. The lysate obtained from transformed E. coli cells containing the rDTB PW8 was clarified by centrifugation and purified by affinity chromatography. The homogeneity of the purified rDTB PW8 was confirmed by immunoblotting using mouse polyclonal anti-diphtheria toxoid antibodies and the immune response induced in animals with rDTB PW8 was evaluated by ELISA and dermonecrotic neutralization assays. The main result of the present study was an alternative and accessible method for the expression and purification of immunogenically reactive rDTB PW8 using commercially available systems. Data also provided preliminary evidence that rabbits immunized with rDTB PW8 are able to mount a neutralizing response against the challenge with toxigenic C. diphtheriae.

Improved Expression of Immunotoxin DT_(386)-GMCSF in E.coli / 中国生物工程杂志

The development of immunotoxin DT386-GMCSF, a fusion protein which bears the N-terminal 386 amino acids of diphtheria toxin and human granulocyte-macrophage colony-stimulating factor (GM-CSF) and targets the GM-CSF receptor (GM-CSFR), has provided a promising alternative therapy to the acute myeloid leukemia (AML). However, the poor expression of the protein in E.coli is still a bottleneck which limits the industrial production. To identify the critical down-regulating factors on the expression of DT386-GMCSF, a series of truncated mutants of DT386-GMCSF at the C-terminal of GM-CSF were generated and expressed in E.coli. The results showed that the encoding sequences for the L114 of the GM-CSF dramatically impact the expression of DT386-GMCSF. On this basis, a serial of mutants integrating amino acid substitutes were generated. The results revealed that the expression level of the mutant DF123GVT, which harbors the amino acids 1-123 of GM-CSF whose L114L115V116 was substituted with G114V115T116, was evidently higher than that of the DT386-GMCSF, whereas the specific cytotoxicity to blast recovered from mice injected with HL60, a cell line highly expresses the GM-CSFR, was similar. These results have provided an important basis for the future development of the immunotoxins targeting the GM-CSFR.

Mutations in E154 of Diphtheria Toxin (DT) and Their Biologic Activity / 生物化学与生物物理进展

According to the results of quantum chemistry calculation and the present research status in the relationship between the structures and the functions of DT, the E154 in DT catalyzing domain was mutated to aspartic acid and arginine in order to study the effects of the alteration on the biological activities. By means of gene site-direct mutation, two mutated genes were prepared and the high performance expression was obtained in E.coli system. The results of toxcity studies indicated that the acute toxicity in guinea pig and cytotoxicities of mutant E154D increased slightly in compared with those of recombination wild toxin, and contrarily, those of E154R decreased obviously.

Conjuncting on targeting toxin of vascular endothelial growth factor a antibody-diphtheria toxin mutation / 中华检验医学杂志

0.05). It is decrease greatly, than those in anti-VEGF control (P0.05).The toxicity of CRM9 control are markedly higher than those in blank control groups (P

pH-Dependent membrane interactions of diphtheria toxin: A genetic approach.

A genetic approach is described for exploring the mechanism by which diphtheria toxin undergoes pH-dependent membrane insertion and transfer of its enzymic A fragment into the cytoplasm of mammalian cells. The cloned toxin expressed in Escherichia coli is secreted to the periplasmic space, where it is processed normally and folds into a native structure. When bacteria synthesizing the toxin are exposed to pH 5, they die rapidly. The toxin undergoes a conformational change that is believed to allow it to be inserted into the bacterial inner membrane and form channels, which proves lethal for the cell. The membrane insertion event mimics the process by which the toxin inserts into the endosomal membrane of mammalian cells, leading to release of the enzymic A fragment into the cytoplasm. The observation of pH-dependent bacterial lethality provides the basis for a positive genetic selection method for mutant forms of the toxin that are altered in ability to undergo membrane insertion or pore formation.

Construction of an Ewing's sarcoma specific expression vector of diphtheria toxin and observation of the killing effect on cultured Ewing's sarcoma cell line / 第三军医大学学报

Objective To construct an expression vector containing Ewing's sarcoma EWS FLI 1 specific binding sequence and diphtheria toxin A chain (DTA) gene sequence to investigate the killing effect of DTA on cultured Ewing's sarcoma cells so as to provide proof for the exploration of new methods for gene therapy for cancer. Methods Expression vector (pS2 DTA) containing EWS FLI 1 specific binding sequence and DTA gene sequence was constructed by replacing the luciferase gene in pS2 with DTA gene at the points of Nco Ⅰ and Xba Ⅰ by molecular technique. After transfection of pS2 DTA into Ewing's sarcoma cells and the control cells, DTA expression and its killing effect on cells were detected. Results pS2 DTA was highly expressed in Ewing's sarcoma cell line, and the killing effect of DTA was much higher than that in the control cells. Conclusion pS2 DTA has selective killing effect on Ewing's sarcoma cells and can inhibit the growth of Ewing's sarcoma cells. So pS2-DTA has potential value in the treatment of Ewing's sarcoma.

Specific expression of the diphtheria toxin A chain gene regulated by the EWS-FLI1 fusion gene in Ewing's sarcoma nude mouse models / 第三军医大学学报

Objective To observe the specific expression of diphtheria toxin A chain gene(DTA) in Ewing's sarcoma nude mice models.Methods pS2DTA plasmid was constructed by the Lus gene in pS2 plasmid was replaced with the DTA gene in pIBI30 plasmid,and transformed into DH5?, then amplified.Five nude mice were injected with 2.4?10~(7) SKES1 cells,sacrificed on week 6,and the tumor tissues were implanted onto the back of another 20 nude mice.Till 6 weeks later the diameter of the tumor up to 0.8-1.0 cm,the Ewing's sarcoma nude mice models were successfully established and randomly assigned to receive the injection of 300 ?g lipidosome+100 ?l normal saline(control group) or 300 ?g lipidosome+100 ?l normal saline+100 ?g pS2DTA (experimental group) once a day for 5 d.On day 4 after the therapy finished,4 nude mice of each group were randomly sacrificed and the DTA expression in the tissue of tumor,liver and myocardium was detected by the immunohistochemistry and RTPCR.Results The immunochemical results showed DTA expression in the tissues of control group was all negative while the DTA expression in the experimental group was negative in the tissues of myocardium and liver,positive in tumor tissue.RTPCR showed DTA expression in control group was negative in all tissues while strongly positive in tumor tissue,weakly positive in liver tissue and negative in myocardiac tissue.Conclusion The pS2DTA regulated by the EWSFLI1 fusion gene has a specific expression in transplanted Ewing's sarcoma in mice.

Effect of diphtheria toxin fragment A on energy coupling in mitochondria. Studies on mouse liver mitoplasts.

The effect of intact diphtheria toxin and of its fragment A on protein synthesis in mouse liver mitoplasts (digitonin-treated mitochondria) was studied. Fragment A inhibited protein synthesis in intact mitoplasts to the same extent as the uncoupler, carbonylcyanide p-trifluoromethoxyphenylhydrazone, but similar effects were not observed in lyzed mitoplasts. Intact diphtheria toxin was without effect in either case. Fragment A strongly stimulated mitochondrial ATPase activity. At concentrations which efficiently inhibited mitochondrial protein synthesis and stimulated ATPase activity, fragment A had no effect on the intramitochondrial concentration of nicotinamide adenine dinucleotides. Moreover, it did not catalyze ADP ribosylation of mitochondrial proteins. The results indicate that the effects observed did not involve the NAD+-glycohydrolase activity of fragment A. [125I]-Labelled fragment A was bound to mitoplasts to about the same extent as the labelled intact diphtheria toxin. The present results suggest that fragment A of diphtheria toxin is capable of inhibiting the energy coupling in mitoplasts, thereby inhibiting protein synthesis. The detailed mechanism of the uncoupling and its possible physiological significance remains to be elucidated.