Elastin-like polypeptides (ELPs) are temperature sensitive biopolymers composed of a Val-Pro-Gly-Xaa-Gly pentapeptide repeat that derived from a structural motif found in mammalian elastin. It was a promising tag for recombinant protein purification. Here, we de novo designed a novel ELPs gene and cloned it into the modified expression vector pET-22b(+). Then, we transformed the recombinant expression vector pET-22b-ELPs into Escherichia coli BL21(DE3). Upon induction by Isopropyl beta-D-Thiogalactoside (IPTG), ELPs was expressed and purified by a non-chromatographic purification method named inverse temperature cycling. The influences of salts types and concentrations on ELPs were also determined. The results showed that the transition temperature of the [KV8F-20] decreased to 19 degrees C by 0.4 mmol/L Na2CO3. Due to its small molecular weight and sensitivity to salt, the ELPs might be a useful purification tag, which can provide a reliable and simple non-chromatographic method for purification of the recombinant protein by inverse transition cycling.
Chromatography ; Elastin ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Peptides ; genetics ; isolation & purification ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Sodium Chloride ; pharmacology

Objective To construct a mammalian vector encoding angiostatin kringle 5 (K5) under the control of ?-fetoprotein (AFP) enhancer and albumin promoter, and to observe the expression of angiostatin by introducting angiostatin gene into hepatocellular carcinoma cells through gene transfection. Methods Angiostatin cDNA was amplified from normal human eukaryotic cells by using RT-PCR. Meanwhile, AFP enhancer and albumin promoter sequences were directed cloned and were inserted into vector pcDNA3.1. The recombinant vector of pcDNA3.1-AFAB-angiostatin K5-His was constructed, which contained the angiostatin K5 cDNA sequence that was under the control of the AFP enhancer and promoter. Angiostatin K5 cDNA was introduced into human AFP positive hepatocellular carcinoma cell lines with the transfected cultured cells that were mediated with Lipofectamine 2000. The expression of angiostatin K5 was analyzed by Western blot and the protein was dectected with anti-His antibody. Results The 500-base pair of angiostatin K5 was in accordance with the expected sequence and the recombinant vector of pcDNA3.1-AFAB-angiostatin K5-His was also confirmed as the anticipated sequence. The expression of angiostatin K5 in AFP positive hepatocellular carcinoma cells was detected both by SDS-PAGE and Western blot. Conclusion Efficient construction and expression of angiostatin K5 to AFP positive cells make it possible for antiangiogenesis therapy of human hepatocellular carcinomas, which may provide a promising approach.

ObjectiveTo study the protective effects of tumor necrosis factor-α(TNF-α) antibody on pancreatic encephalopathy in rats. MethodsSixty SD rats were randomly divided into the normal control group, acute necrotizing pancreatitis induction group and TNF-α antibody treated group. Acute hemorrhage necrotizing pancreatitis model in rats were induced by retrograde injection of 5% sodium taurocholate into the pancreatobiliary duct. Serum TNF-α was detected and animals were killed 12 h after drug administration. The changes of brain water contents, leucocyte accumulation and adhesion were measured, and pathological studies of pancreas and brain were performed. ResultsIn group of TNF-α antibody treated, serum TNF-α level decreased, brain water contents and leucocytes accumulation and adhension decreased significantly than that of acute necrotizing pancreatitis induction group (P<0.05). The histopathological change of pancrea was alleviative. ConclusionTNF-α antibody can alleviate the brain damage in acute hemorrhage necrotizing pancreatitis.

Objective To investigate the expression of tumor associated glycoprotein-72 (TAG-72) in human breast cancer cells. Methods Two breast cancer cell lines MCF-7 and Bcap-37 were cultured and prepared. TAG-72 expressions in MCF-7 and Bcap-37 were detected immunochemically with anti-TAG-72 single chain variable fragment (scFv). Results TAG-72 was positively expressed in MCF-7 cells but negatively expressed in Bcap-37 cells. Conclusion Tumor associated antigen TAG-72 is expressed in certain human breast cancer cells, which indicate TAG-72 may be used as a tumor maker and anti-TAG-72 scFv may play a role in the diagnosis and treatment of breast cancer.

Objective To study the role of down regulation of bcl 2 expression of human cholangiocarcinoma cells with bcl 2 mRNA cleaving ribozyme (RZ). Methods Synthetic gene of bcl 2 mRNA cleaving RZ was recombined with the expression vector of eukaryotic cells. The recombined vector was used to transfect human cholangiocarcinoma cells. The expression of bcl 2 in human cholangiocarcinoma cells was observed by immunocytochemistry and flow cytometry. Results The expression of bcl 2 in human cholangiocarcinoma cells transfected with RZ gene was lower than that of control group. Spontaneous apoptosis was observed in a few of the cells. Conclusion bcl 2 mRNA cleaving RZ can down regulate the expression of bcl 2 of cholangiocarcinoma cells notably and can also induce spontaneous apoptosis.

Objective　To investigate the relationship between canceration and primary operation mode for congenital choledochal cysts(CCC). Methods　The clinical data of 21 patients with CCC treated in the last 30 years were analysed retrospectively. Results　In this series, the incidence of carcinoma was 14.8%; the canceration rate after internal drainage operation was significantly higher than that after resection of the cyst(P<0.001); the age of canceration after internal drainage was younger than that of resection of the cyst(P<0.01) and patients without operation(P<0.01); the interval time of canceration after internal drainage operation was significantly less than that of resection of the cyst(P<0.01); the age of carcinoma would be 15.4 years younger in internal drainage operation patients than that in patients without operation. Conclusions　Internal drainage, which could accelerate the occurrence of canceration of CCC, should be abandoned; resection of the cyst is recommended as the therapy of the first choice.

Purpose: To explore the autocrinism of vascular endothelial growth factor ( VEGF) in human hepatocellular carcinoma cell and its significance. Methods: The expression of VEGF receptors was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) in human hepatocellular carcinoma cell lines SMMC7721, HHCC and HepG2 in vitro. The expression of VEGF in human hepatocellular carcinoma cells was inhibited by synthetic antisense oligodeoxynucleotide (ODN). Phenotypic alterations in human hepatocellular carcinoma cells by antisense reduction of VEGF expression were observed. And the alterations for expression of VEGF receptors in human hepatocellular carcinoma cell membranes were analyzed by flow cytometry. Results: Flt-1 was expressed faintly in SMMC-7721 cell whereas KDR was expressed in HHCC cell and HepG2 cell in vitro. Compared to control, antisense ODN against VEGF inhibited HHCC cell proliferation in vitro in a dose-dependent manner and had no effects on SMMC-7721 cell and L929 cell. When the concentration of antisense ODN was 2.5 ?mol/L,5 ?mol/L and 10 ?mol/L, the proliferation of HHCC cell was inhibited by 26%, 41 % , and 50% respectively. Compared with control, flow cytometry analysis showed a decrease in cell number in S phase and a pre-apoptosis peak in HHCC cell treated by antisense ODN. There was no alteration of cell cycle in SMMC-7721 cell. The positive rate of KDR expression in HHCC cell membrane was 19. 2%, 29. 8% and 31. 2% in the antisense ODN, sense ODN and control groups, respectively. The expression of KDR in HHCC cell membrane was markedly inhibited by antisense ODN against VEGF compared with the control group, as measured by flow cytometry. There was no alteration for expression of Flt-1 in SMMC-7721 cell membrane. Conclusions: VEGF produced by human hepatocellular carcinoma cells acts directly upon human hepatocellular carcinoma in an autocrinal manner and may promote the proliferation and differentiation of human hepatocellular carcinoma cells via the activation of the KDR-dependent signaling pathway.

ObjectiveTo investigate the expression of MUC1 in thyroid cancer and benign thyroid diseases (nodular goiter and adenoma) and its clinical significance. Methods The expression level of MUC1 was examined by immunohistochemical analysis with MUC1 monoclonal antibody in 68 samples of thyroid carcinoma and in 18 samples of benign diseases and 10 normal thyroid tissues. Results Immunohistochemical analysis showed positive expression of MUC1 in 51 out of 68 cases of thyroid cancer, in 4 out of 18 cases of benign thyroid diseases and 1 out of 10 normal control thyroid tissue, with a difference statistically significant between the malignancy and benign and normal control (? 2=17.20,P0.05).Conclusion The over-expression of MUC1 in thyroid cancer may act as diagnostic markers of thyroid carcinoma.

Objective To investigate the expression of MUC1 in primary liver carcinoma (PLC) and in cirrhotic liver tissue and its clinical significance in the diagnosis and immuotherapy of PLC. Methods The expression of MUC1 was examined by immunohistochemical analysis in 43 samples of primary liver carcinoma (PLC) , incluing 34 samples of hepatocellular carcinoma (HCC), 9 samples of cholangiocarcinoma (CC) ;and 12 samples of cirrhotic liver tissue and 6 samples of normal liver tissues. Results Immunohistochemical analysis showed over- expression of MUC1 in PLC , which aberrantly localized in the cancer cell membrane; while expression of MUC1 was detected only in 2 cirrhotic liver samples,and no expression in normal liver tissue. The expressional level of MUC1 in PLC was significantly higher than that in cirrhotic and normal liver tissue ( P 0.05),but the expressional levels between with portal cancer emboli group and without portal cancer embloli group was significant difference( P

Objective To study the protective effects of ulinastatin and tumor necrosis factor-?(TNF-?) antibody on ischemia and reperfusion injury of liver in rats. Methods One hundred and twenty male SD rats were randomly divided into four groups: the normal control group, ischemia and reperfusion group, TNF-? antibody group and ulinastatin plus TNF-? antibody group. And the animals were killed after 60 minutes ischemia of liver followed by reperfusion for 1,3,6 and 12 hours. Serum alanine aminotransferase(ALT) and malondialdehyde(MDA) were detected, and liver histopathologic lesions were observed. Results After ischemia and reperfusion, the serum level of ALT and MDA remarkedly increased, and the hepatic congestion was prominent. Treatment of ulinastatin and TNF-? antibody could decrease the serum level of ALT and MDA significantly, and relieve hepatic congestion. Conclusions Ulinastatin and TNF-? antibody can suppress the inflammatory reaction induced by hepatic ischemia and reperfusion, and has protective effects on rat hepatic ischemia and reperfusion injury.