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 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.

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.

Aim To investigate the effect of intrauterine injection of rat fetal hepatocytes on homograft rejection reaction. Methods The skin graft from a male LOU/CN rat was transplanted to a female recipient CHN rat of 7 to 9 weeks after parturition, and then survival time of the graft was observed. Simultaneously, homograft rejection reaction was examined by mixture lymphocyte culture. Results As compared with control group, survival time of transplanted skin graft was obviously prolonged. Mixure lymphocyte culture demonstrated that homograft rejection reaction was inhibited markedly. Conclusion Intrauterine injection of rat fetal hepatocytes wuld obviously inhibite homograft rejection reaction, thus prolonging survival time of the graft.

Aim To study relationship between expressions of apoptosis-related gene bax and bcl-x in heopatocellular carcinoma (HCC) tissues and its clinical feature. Mothods The expression of Bax and Bcl-x were determined by immunohistochemistry with EnvisionTM system. Results Bcl-x and Bax proteins were detectable in 20 and 19 out of 40 HCCs, respectively. Their expressions in HCC cells were lower than those in cirrhosis(P＜ 0.05), The expression of Bax and Bcl-x in highly or intermediatly differentiated HCC cells were highed than that in low-diffe-rehtiated HCC cells suggesting that there was significant relationship among bcl-x and bax expressions and differential degree， clinical stages of HCC cells， and AFP level. However, the expression of this proteins showed no relationship with patient's sex and age(＞ 60 or ＜ 60 years old) (＞ 0.05). Conclusion Apoptosis-related genes bcl-x and bax participate partially in apoptotic regulation of HCC cells, and show that their expressions have correlation with differential degree, clinical stage of HCC cells and AFP levels.

Aim To investigate the expression of vascular endothelial growth factor(VEGF) and its receptors in human hepatocellular carcinoma cell lines SMMC7721, HHCC and HepG2. Methods Immunohistochemical staining and reverse-transcriptase polymerase chain reaction(RT-PCR) were used to detect the mRNA and expression of VEGF and its receptors: VEGF-R1(Flt-1) and VEGF-R2 (KDR) in three human hepatocellular carcinoma cell lines, SMMC7721, HHCC and HepG2, as compared with ECV304 cells(human umbilical vein endothelial cells) and L929 cells(mouse fibroblast). Results All three human hepatocellular carcinoma cell lines expressed VEGF protein. Flt-1 mRNA and protein could be detected in SMMC7721 cells while KDR in HHCC and HepG2 cells. Conclusion The expressions of Flt-1 and KDR suggests that VEGF may be an autocrine growth factor for human hepatocellular carcinoma, at least for cell lines in vitro.