ABSTRACT
K5 polysaccharide of high molecular weight (HLW) can be splitted into low molecular weight (LMW) K5 polysaccharide by K5 lyase which can be used as the substrate of partial synthesis low molecular heparin sulfate (HS). To prepare recombinant K5 lyase (Elma) and analyze its biological activity. The gene of Elma was cloned by PCR amplification and was ligated with pET-28a. Then the recombinant expression vector pET-28a-Elma was transformed into Escherichia coli BL21 (DE3). After induction with 0.2 mmol/L IPTG at 16 degrees C for 5 h, Elma was successfully expressed, SDS-PAGE analysis demonstrated that the enzyme constituted more than 30% of the total cell proteins. After Ni(2+)-NTA affinity and G-75 chromatography, the purity of enzyme was more than 95%. Enzymatic activity was determined according to the change of absorbance at 232 nm per ml of the sample. The reduction of the polysaccharide molecular weight could be detected by PAGE electrophoresis. Elma can partially split HA and HS. Its optimal reatcion temperature is 37 degrees C and the optimal reaction pH is 7.0.
Subject(s)
Bacterial Capsules , Metabolism , Escherichia coli , Genetics , Genetic Vectors , Genetics , Heparin , Metabolism , Lyases , Genetics , Metabolism , Recombinant Proteins , GeneticsABSTRACT
【Objective】 To study the location effect of the Mc Ab G9 to human esophageal carcinoma in tumor bearing nude mice. 【Methods】 125 I-G9 was prepared by Chloramine-T method. The distribution of 125 I-G9 was detected at different time (24, 48, and 72 h) after peritoneal injecti on. The percentage of the injected dose per gram of tissue(%ID/g) and the ratio of Tumor/non-Tumor were calculated. 【Results】 The distribution of 125 I -G9 in tumor at the third day was showed by autoradiography obviously higher th an in other organ/tissue (except blood) and the highest is at the 48 h. The T/NT values are 2-7. The autoradiography indicated that radioactivity concentrated in tumor tissue. The concentration in tumor edge is more obvious than in the ce nter. 【Conclusion】 125 I-G9 has a considerable targeting activity and can well locate in esophageal carcinoma tissue in tumor bearing nude mice.