Objective The TCS gene without N-and C-terminal sequences was cloned into pET-29b in order to get the recombinant trichosanthin(rTCS) at the relatively high level in E.coli,and the rTCS immunogenicity was compared with natural TCS(nTCS). Methods TCS gene without the N-and C-terminal coding sequences was amplified by RT-PCR,and then inserted into pET-29b.The soluble rTCS was induced with IPTG and purified through metal chelate affinity chromatography,the purity and content were showed by the thin-layer scan analysis and bradford assay.After the rabbits were immunized with purified rTCS and nTCS,respectively,the titer and specificity of polyclonal antibody were detected by indirect ELISA assay and Western blotting. Results 1.The vector pET-29b-TCS was constructed successfully after it was confirmed with the same restriction enzymes.2.The rTCS attained the maximum in the presence of 1 mmol/L IPTG for 4?h at 30℃.3.The soluble rTCS was expressed at relatively high level(36% of total protein in E.coli) and the yield of rTCS with a purity of 95% was 1.2?g in 1 L bacterium.4.The polyclonal antisera titer of nTCS was significantly higher than that of rTCS(1∶40?960 and 1∶5?120 respectively).5.The specific reaction between rTCS and anti-nTCS antibody was detected by Western blotting.Conclusion The high level expressing vector pET-29b-TCS,carrying TCS gene withoug the N-and C-terminal coding sequences,was constructed successfully.The immunogenicity of rTCS was remarkably lower than that of nTCS,which indicates that glycosylation of nTCS has a relationship with the antigenicity.

AIM: To further study the effect of Ershen Zeshu Mixture, a Chinese medicine, on the peritoneal lymphatic stomata and the ascites drainage in experimental mice model of CCl 4-induced fibrosis.By the application of NO supplier (sodium nitroprusside,SNP) and NO suppressor (N G-mononethyl-L-arginine,L-NMMA),the effect of NO on the peritoneal lymphatic stomata and the ascites drainage was studied to clarify the relationship between Ershen Zeshu Mixture and NO in the regulation of the peritoneal lymphatic stomata.METHODS: The mouse model of fibrosis was established with the application of intragastric installations of carbon tetrachloride one time every three days;Scanning electron microscope and computer image processing were used to detect the girth,area and distribution density of the peritoneal lymphatic stomata;the concentration of urinary ions was measured in the experiment.RESULTS: ①Compared to NS group and model group,Ershen Zeshu Mixture could significantly increase the girth and the area of the peritoneal lymphatic stomata,promote its distribution density ( P

Objective To investigate the relationship between the polymorphisms of both aldose reductase (AR) and endothelial nitric oxide synthase (eNOS) genes in chromosome 7q35 and the susceptibility to diabetic nephropathy (DN) in type 2 diabetes of Han nationality in North China. Methods The C(-106)T in the promoter region of AR gene as well as the G894T in exon 7 and the 27 bp repeat polymorphism in intron 4 of the eNOS gene were investigated in 85 healthy controls and 134 type 2 diabetics with or without DN. The C(-106)T as well as the G894T genotype were determined by PCR RFLP method and sequencing the PCR products. The 27 bp repeat polymorphism alleles were determined by PCR combined with agarose gel electrophoresis and sequencing the PCR products. Results The frequencies of the C allele and C/C genotype in AR gene as well as T allele and T/G genotype in exon 7 and the 4a allele and 4a/4b genotype in intron 4 of eNOS gene were significantly higher in DN+ group than those in DN-group (all P