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 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 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 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.

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.

Objective　To investigate whether or not the antisense oligodeoxynucleotide (ODN) of vascular endothelial growth factor (VEGF) can supresses the protein expression of VEGF.Methods　SMMC 7721 is a cell line that expresses VEGF.Therefore the medium conditioned by SMMC 7721 stimulates proliferation of bovine aortic endothelial cell (BAEC).Synthesized antisense ODN complementary to one region of VEGF mRNA was added into the medium of SMMC 7721.The expression of VEGF and the stimulation effect of SMMC 7721 was then determined.Result　It was found that by means of inhibition of VEGF expression,the stimulation effect of SMMC 7721 was inhibited by the VEGF antisense ODN in a dose-dependent manner.At a dosage of 1,2.5,5,10μmol/L,the inhibition rate was 63.2%,82.4%,210% and 287.5%,respectively.Conclusion　It is promising that VEGF antisense ODN works as a new gene therapeutic modaliey for antiangiogenesis of HCC.

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.

To elucidate the effect of p27 KIP1 on cell cycle and proliferation of gastric carcinoma cells, we transfected the full e length cDNA of p27 KIP1 into human SGC7910 gastric cancer cells by the method of lipofectin transfection. Expression of p27 KIP1 at protein or mRNA level was analyzed by Western blotting, and RNA dot blotting, respectively. Effect of p27 KIP1 on cell growth was observed by trpan blue exclusion assay and anchorage independent growth in soft agar. Flow cytometry was applied to assess the effect of p27 KIP1 on cell cycle. The results showed that the expression of p27 KIP1 at protein or mRNA level increased evidently in SGC7901 cells transfected with p27 KIP1 . The cell growth was reduced by 42% 48h post induction with zinc as determined by cell viability assay. The rate of anchorage independent growth in soft agar decreased significantly. p27 KIP1 over expression caused cell arrest at G 1 by 36%(from 33 68% to 69 29%, P