AIM To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')2 fragment of mAb HAbl8 against human hepatocellular carcinoma (HCC), and identify its activity in ...AIM To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')2 fragment of mAb HAbl8 against human hepatocellular carcinoma (HCC), and identify its activity in order to use SAg in the targeting therapy of HCC.METHODS MAb HAbl8 was extracted from the abdominal dropsy of Balb/ c mice, and was purified through chromatography column SP-40HR with Fast protein liquid chromatography (FPLC) system. The F(ab')2 fragment of mAb HAb18 was prepared by papainic digestion method. The conjugate of mAb HAb18 F(ab')2fragment and SEA was prepared with chemical conjugating reagent N-succinimidyl-3-( 2-pyridyldithio) propionate (SPDP) and purified through chromatography column Superose 12with FPLC system. The molecular mass and purity of each collected peak were identified with SDS-PAGE assay. The protein content was assayed by Lowry's method. The antibody activity of HAb18 F (ab')2 against HCC in the conjugate was identified by indirect immunocytochemical ABC method, and the activity of SEA in the conjugate to activate peripheral blood mononuclear cells (PBMC) was identified with MTT assay.RESULTS The lgG mAb HAb18 was extracted,and purified successfully. Immunocytochemical staining demonstrated that it reacted with most of HHCC cells of human HCC cell line. There were two peaks in the process of purification of the prepared HAb18 F(ab)2-SEA conjugate. SDS-PAGE assay demonstrated that the molecular mass of the first peak was about 130 ku, and the second peak was the mixture of about 45 ku and a little 100 ku proteins. The immunocytochemical staining was similar in HAb18 F (ab ')2-SEAconjugate and HAb18 F (ab ')2, i.e., thecytoplasm and/or cell membranes of most HHCC cells were positively stained. The MTT assay showed that the optical absorbance (A) value at 490 nm of HAb18 F (ab')2-SEA conjugate was 0.182 ± 0.012, that of negative control was 0.033± 0.009, and there was significant difference between them ( P < 0.05).CONCLUSION SPDP is a good proteinconjugating reagent and can 展开更多
The effect of enterotoxins is to induce the production of endogenous IF. St. aureus enteropathogenic proteins (enterotoxins) possess an antitumour effect. After intraperitoneal inoculation, they decrease the size and,...The effect of enterotoxins is to induce the production of endogenous IF. St. aureus enteropathogenic proteins (enterotoxins) possess an antitumour effect. After intraperitoneal inoculation, they decrease the size and, in some cases, prevent the development of the human hypernephroma in the cheek pouch of golden hamsters. The effect of enteropathgenic proteims may possibly consist in inducing the production of endogenous immune interferon which activates the host immune system and enhances the rejection of heterologous tumour cells.展开更多
Staphylococcus aureus represents a public health challenge all over the world. Therefore, this study aims to analyze the prevalence of five genes (sea, seb, sec, see and seg) encoding the staphylococcal enterotoxins i...Staphylococcus aureus represents a public health challenge all over the world. Therefore, this study aims to analyze the prevalence of five genes (sea, seb, sec, see and seg) encoding the staphylococcal enterotoxins in S. aureus isolated from different sources and to evaluate the association of these toxins in comparison to susceptibility towards 12 antimicrobials;antimicrobial susceptibility was conducted by disc diffusion method. Detection of staphylococcal enterotoxins was performed by PCR and the ability to express these genes was assessed among isolates by RT-PCR. The most common enterotoxin gene was sea gene (66%), followed by seb, sec, see and seg (38%, 23%, 19% and 5%) respectively. Expression of sea, seb and seg genes was variable. However, sec and see genes were not expressed by any of the tested isolates. No statistically significant association exists between (seb, sec and see) and isolation sources, while the sea was significantly associated with clinical isolates. High significant correlation was found between elevated sea expression and multidrug-resistance. Our findings indicate that the pathogenic potential of S. aureus may be greater than previously thought. This emphasizes the utmost need to implement proactive measures and more emphasis will be placed on the application of hygiene practices in hospitals to control S. aureus infection and enterotoxins production.展开更多
基金Supported by the National 863 Project of China,No.102-09-01-02National Natural Science Foundation of China,No.39770827
文摘AIM To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')2 fragment of mAb HAbl8 against human hepatocellular carcinoma (HCC), and identify its activity in order to use SAg in the targeting therapy of HCC.METHODS MAb HAbl8 was extracted from the abdominal dropsy of Balb/ c mice, and was purified through chromatography column SP-40HR with Fast protein liquid chromatography (FPLC) system. The F(ab')2 fragment of mAb HAb18 was prepared by papainic digestion method. The conjugate of mAb HAb18 F(ab')2fragment and SEA was prepared with chemical conjugating reagent N-succinimidyl-3-( 2-pyridyldithio) propionate (SPDP) and purified through chromatography column Superose 12with FPLC system. The molecular mass and purity of each collected peak were identified with SDS-PAGE assay. The protein content was assayed by Lowry's method. The antibody activity of HAb18 F (ab')2 against HCC in the conjugate was identified by indirect immunocytochemical ABC method, and the activity of SEA in the conjugate to activate peripheral blood mononuclear cells (PBMC) was identified with MTT assay.RESULTS The lgG mAb HAb18 was extracted,and purified successfully. Immunocytochemical staining demonstrated that it reacted with most of HHCC cells of human HCC cell line. There were two peaks in the process of purification of the prepared HAb18 F(ab)2-SEA conjugate. SDS-PAGE assay demonstrated that the molecular mass of the first peak was about 130 ku, and the second peak was the mixture of about 45 ku and a little 100 ku proteins. The immunocytochemical staining was similar in HAb18 F (ab ')2-SEAconjugate and HAb18 F (ab ')2, i.e., thecytoplasm and/or cell membranes of most HHCC cells were positively stained. The MTT assay showed that the optical absorbance (A) value at 490 nm of HAb18 F (ab')2-SEA conjugate was 0.182 ± 0.012, that of negative control was 0.033± 0.009, and there was significant difference between them ( P < 0.05).CONCLUSION SPDP is a good proteinconjugating reagent and can
文摘The effect of enterotoxins is to induce the production of endogenous IF. St. aureus enteropathogenic proteins (enterotoxins) possess an antitumour effect. After intraperitoneal inoculation, they decrease the size and, in some cases, prevent the development of the human hypernephroma in the cheek pouch of golden hamsters. The effect of enteropathgenic proteims may possibly consist in inducing the production of endogenous immune interferon which activates the host immune system and enhances the rejection of heterologous tumour cells.
文摘Staphylococcus aureus represents a public health challenge all over the world. Therefore, this study aims to analyze the prevalence of five genes (sea, seb, sec, see and seg) encoding the staphylococcal enterotoxins in S. aureus isolated from different sources and to evaluate the association of these toxins in comparison to susceptibility towards 12 antimicrobials;antimicrobial susceptibility was conducted by disc diffusion method. Detection of staphylococcal enterotoxins was performed by PCR and the ability to express these genes was assessed among isolates by RT-PCR. The most common enterotoxin gene was sea gene (66%), followed by seb, sec, see and seg (38%, 23%, 19% and 5%) respectively. Expression of sea, seb and seg genes was variable. However, sec and see genes were not expressed by any of the tested isolates. No statistically significant association exists between (seb, sec and see) and isolation sources, while the sea was significantly associated with clinical isolates. High significant correlation was found between elevated sea expression and multidrug-resistance. Our findings indicate that the pathogenic potential of S. aureus may be greater than previously thought. This emphasizes the utmost need to implement proactive measures and more emphasis will be placed on the application of hygiene practices in hospitals to control S. aureus infection and enterotoxins production.
