[Construction and expression of recombinant human serum albumin-EPO fusion protein].

UNLABELLED: OBJECTIVE To construct the recombinant plasmid pCI-HLE encoding human serum album-EPO (HSA-EPO) fusion protein and to express it in CHO cell. METHODS: The cDNA encoding human serum album and EPO were amplified by PCR, and then spliced with the synsitic DNA fragment encoding GS (GGGGS), by overlap PCR extension to form LEPO. After BamH I digestion, the HSA and LEPO was ligated to generate the fusion HSA-EPO gene and was then cloned into the expression vector pCI-neo to generate the recombinant plasmid pCI-HLE. The plasmid pCI-HLE was transfected into CHO cell by liposome protocol. Then, the recombinant cells were screened by G418 and identified by PCR and Western blot. Expression of fusion protein was evaluated by Enzyme Linked Immunosorbent Assay (ELISA). RESULTS: Restrictive enzymes digestion and DNA sequencing revealed that HSA-EPO fusion gene was cloned into expression vector pCI-neo successfully. PCR and Western blot analysis confirmed that the fusion gene was integrated in the genome of CHO cells and expressed successfully. The HSA-EPO production varied from 86 Iu/(mL x 10(6) x 72 h) to 637 IU/(mLx 10(6) x 72 h). CONCLUSION: The results confirmed that HSA-EPO fusion gene can be expressed in the CHO cells, with EPO immunogenicity, which could serve as foundation for the development of long-lasting recombinant HSA-EPO protein.

[Establish efficiently transformation system of the Isatis indigotica].

OBJECTIVE: To build up a method of efficiently transforming Isatis indigotica with the Agrobacterium. METHODS: Two kinds of Agrobocterium: ATCC 15834 and RI1601 were used to treat different fraction of Isatis indigotica. Explored the effect of cocultured, different antibiotic concentration and the media on multiply the hairy roots. RESULTS: The explants with out coculture had had higher survival rate rooting rate and earlier sprout time. After ultrasonic treatment of plant, the indacement rate was two times than that of untreated one; The best antibiotic concentration was 400 mg/L; The proportion of the grow speed of the hairy root in the liquid culture media was 2 - 3 times than that of solid culture media, and 37 times of common roots. CONCLUSION: The method is useful for establishing an efficiently transformatiem system of Isatis indigotica by Agrobacterium.

Zinc finger protein A20 promotes regeneration of small-for-size liver allograft and suppresses rejection and results in a longer survival in recipient rats.

BACKGROUND: Small-for-size liver allografts without immunosuppression have decreased survival compared with full-for-size grafts for the concomitant regeneration-induced accelerated rejection. This study was designed to examine the effect of zinc finger protein A20 on liver allograft regeneration and acute rejection using a high responder rat model (DA-->Lewis) of 30% partial liver transplantation. MATERIALS AND METHODS: Adenovirus carrying the full length of A20 was introduced into liver grafts by ex vivo perfusion via the portal vein during preservation, physiological saline (PS), and empty Ad vector rAdEasy served as controls; then small-sized liver transplants were performed. Liver graft regeneration was assessed, as well as graft rejection, hepatocyte apoptosis, nuclear factor kappa B activation, and intercellular adhesion molecule-1 mRNA expression in liver graft sinusoidal endothelial cells (LSECs), infiltration of liver graft infiltrating mononuclear cells (LIMCs), and the subproportion of NK and NKT cells, activity of liver graft NK-like cells, interferon gamma (IFN-gamma) production, and animal survival. RESULTS: Ex vivo transfer of the A20 gene resulted in overexpression of A20 protein in LSECs and hepatocytes 24 h after partial liver transplantation. Regeneration of the small-sized liver allograft was augmented by A20 overexpression, the DNA synthesis of hepatocytes on d 4 post-transplant was increased in A20 group compared with PS and rAdEasy groups (P < 0.01). Hepatocyte apoptosis was inhibited by A20 (P < 0.001). On d 4 after transplantation, histological examination revealed a more exiguous cellular infiltration and mild rejection in A20 group but a more vigorous cellular infiltration in the sinusoidal area and more severe rejection in PS and rAdEasy group. Nuclear factor kappa B activation and intercellular adhesion molecule-1 mRNA expression in LSECs were suppressed by A20 overexpression. Flow cytometry analysis showed a marked down-regulation of LIMCs number by A20, including more prominent decrease in the subproportion of NK and NKT cells. Activity of liver graft NK-like cells, IFN-gamma mRNA expression in LIMCs, and serum IFN-gamma protein level were also suppressed by A20 overexpression (P < 0.05), respectively. Survival days of A20 rats were longer than those of PS rats and rAdEasy rats (P < 0.01), whereas survival days of rAdEasy rats were shorter than those of PS rats (P < 0.01). CONCLUSIONS: These data suggest that A20 overexpression could effectively promote small-sized liver allograft regeneration, suppress rejection, and prolong survival of recipient rat. These effects of A20 could be related to an inhibition of LSECs activation, suppression of infiltration of LIMCs, and the subpopulations such as NK and NKT cells into liver graft, and inhibition of hepatocyte apoptosis.

Construction of the recombinant adenovirus vector carrying antisense multidrug resistance gene.

BACKGROUND: Multidrug resistance proteins serve as transporters for chemical drugs in human malignancies. The objective of this study was to construct a homologous recombinant adenovirus carrying a reversal fragment of multidrug resistance gene 1 (mdr1) gene cDNA sequence. METHODS: The fragment of the mdr1 gene from the plasmid pHaMDR1-1 carrying the whole human mdr1 cDNA sequence was inserted reversely into the shuttle plasmid pAdTrack-CMV of adenoviral vector system AdEasy. The homologous recombination process was taken place in E. coli BJ5183 with the backbone plasmid pAdEasy-1. After packaging in 293 cells, recombinant adenoviral plasmid was generated. The recombinant adenoviral plasmid was identified by polymerase chain reaction (PCR), restriction endonucleases digest, DNA sequence analysis and fluorescence microscopic photograph, respectively. RESULTS: The recombinant adenovirus pAdEasy-GFP-ASmdr1 was successfully constructed and identified by PCR, restriction digest, and sequencing with strong green fluorescence expression in fluorescence microscopic photograph. CONCLUSIONS: The recombinant adenoviral mdr1 vector would introduce the antisense mdr1 gene into the human multidrug resistance hepatocellular cell line effectively, which would provide an experimental basis to study the multidrug resistance in human hepatocellular carcinoma.

Effects of multidrug resistance, antisense RNA on the chemosensitivity of hepatocellular carcinoma cells.

BACKGROUND: Multidrug resistance is a major obstacle in cancer chemotherapy. We examined whether the antisense RNA of multidrug resistance gene 1 (mdr1) could reverse multidrug resistance in the human hepatocellular carcinoma (HCC) cell line SMMC7721/ADM. METHODS: The recombinant adenoviruses pAdEasy-GFP-ASmdr1 product was produced by the adenoviral vector AdEasy system, which can express antisense RNA against the mdr1 gene. Following that, the recombinant adenovirus was transfected into the P-glycoprotein-producing multidrug resistance cell line, SMMC7721/ADM human HCC cells resistant to adriamycin (ADM) and daunorubicin (DNR). In order to investigate the reversal of multidrug resistance phenotype, we measured the expression of mdr1 mRNA by RT-PCR and the production of P-glycoprotein by flow cytometry. The sensitivities for ADM and DNR SMMC7721/ADM cells were examined by [3-(4, 5-dimethylthi-azol-2-yl)-2,5 diphenyl-terazolium bromide] (MTT) analysis. RESULTS: The low-level expression of mdr1 mRNA and P-glycoprotein production were observed in parental sensitive cells SMMC/7721 in addition to the overexpression of mdr1 mRNA and P-glycoprotein in SMMC7721/ADM cells. The transfection of antisense-RNA into SMMC7721/ADM cells resulted in decreases of mdr1 mRNA and P-glycoprotein, but increase of drug sensitivities. The sensitivities of transfected SMMC7721/ADM cells to ADM and DNR in IC50 reduced by 31.25% and 62.96% respectively. CONCLUSIONS: Mdr1 antisense RNA can increase the sensitivities of SMMC7721/ADM cells to anticancer drug by decreasing the expression of the mdr1 gene and inhibiting P-glycoprotein expression. This strategy may be applicable to cancer patients with P-glycoprotein mediated multidrug resistance.

[Recombinant adenovirus vectors carrying antisense matrix metalloproteinase-2 inhibits hepatocellular carcinoma growth in vivo].

OBJECTIVE: To investigate the inhibitory effect of a recombinant adenoviral vector carrying antisense matrix metalloproteinase-2(MMP2) on the growth of hepatocellular carcinoma(HCC) in vivo. METHODS: The recombinant adenoviral vector carrying antisense MMP2(Ad-MMP2(AS))which had been constructed by us in readiness was used to infect the human HCC cell line (Bel-7402). Then the invasiveness of the Bel-7402 cells was assayed in Matrigel, and the production of MMP2 in the Bel-7402 cells was detected with Western blot analysis and Gelatin zymography. After the Ad-MMP2(AS)-infected Bel-7402 cells being subcutaneously inoculated in nude mice, the production of tumors was under observation, and then Ad-MMP2(AS) was injected intratumorally into the pre-existing tumors. RESULTS: Compared with PBS or Ad-CMV-infected cells, infection of Bel-7402 cells with Ad-MMP2(AS) significantly reduced MMP2 enzyme activity, the invasiveness resulted in 52% reduction in Matrigel assays, and the tumor volume displayed a 4.3-fold reduction in nude mice. In addition, direct intratumoral injection of Ad-MMP2(AS) into pre-existing tumors significantly impaired the further expansion of the tumor mass and resulted in a 63% reduction in tumor cell growth. CONCLUSION: The recombinant adenovirus with antisense MMP2 can effectively inhibit the invasiveness and growth of Bel-7402 cells in vitro and in vivo, and has a therapeutic potential for HCC.

[Reversal of the multidrug resistance in human hepatocellular carcinoma cells by recombinant adenoviral delivery of the MRP antisense RNA in vitro].

OBJECTIVE: To explore the effect of multidrug resistance-associated protein (MRP) antisense RNA mediated by recombinant adenoviruses on reversal of the multidrug resistance in human drug-resistant hepatocellular carcinoma (HCC) cells. METHODS: The HCC cells were first treated in vitro by the recombinant adenoviruses containing antisense MRP. Chemosensitivity of all of the cells to ADM and DNR was determined by MTT assay. The levels of the MRP mRNA and its product P190 were measured by RT-PCR and flow cytometry (FCM) respectively according to the time after transfection. The accumulation of the DNR in these cells was analyzed by FCM simultaneously. RESULTS: IC50 of the cells infected by Ad-Asmrp to ADM and DNR was 0.487 microg/ml and 0.328 microg/ml respectively, with the factors of resistance (RF) decreased by 36.8 and 35.4 respectively (P < 0.05). Continuous decrease in levels of MRP mRNA and P190 was observed 24 h and 48 h after infection respectively (P < 0.01). The intracellular DNR accumulation was increased simultaneously in the Ad-Asmrp transfectants (P < 0.001). CONCLUSIONS: MRP antisense RNA mediated by recombinant adenoviruses could increase chemosensitivity and partially reverse the MDR in HCC cells in vitro by inhibit expression of MRP gene. The study would provide experimental basis for the reversal of MDR in HCC by antisense RNA technology mediated by recombinant adenoviruses.

[Inhibition of adenovirus-mediated gene transfer of antisense matrix metalloproteinase-2 on hepatocellular carcinoma growth in vivo].

OBJECTIVE: To investigate if a recombinant adenoviral vector carrying antisense matrix metalloproteinase-2 (MMP2) gene would inhibit the growth of hepatocellular carcinoma (HCC) in vivo. METHODS: Using the recombinant adenoviral vector carrying antisense MMP2 gene (Ad-MMP2AS) which was constructed by us previously, to infect the human HCC cell line (Bel-7402). Then the invasiveness of the Bel-7402 cells was assayed in Matrigel, and the production of MMP2 in the Bel-7402 cells was detected with Western blotting analysis and Gelatin zymography. Then the Ad-MMP2AS-infected cells were subcutaneously inoculated in nude mice. After tumors developed, Ad-MMP2AS was injected intratumorally into pre-existing tumors. The tumors were removed, sectioned, and stained with H E. RESULTS: Compared with PBS or Ad-CMV-infected cells, the infected Bel-7402 cells with Ad-MMP2AS injections significantly reduced their MMP2 enzyme activity and invasiveness about 52.05% in Matrigel assays, and the tumor volumes in nude mice resulted in a 3.3-fold reduction. In addition, direct intratumoral injection of Ad-MMP2AS into pre-existing tumors significantly prevented further expansion of the tumor masses and resulted in a 63.06% reduction in tumor cell growth. CONCLUSION: The recombinant adenovirus with antisense MMP2 can effectively inhibit the invasiveness and growth of Bel-7402 cells in vitro and in vivo, and it has a therapeutic potential for HCC.

[Recombinant adenovirus vectors carrying antisense MMP2 inhibit invasion of HCC cells in vitro].

OBJECTIVES: To construct a recombinant adenoviral vector carrying antisense matrix metalloproteinase-2 (MMP2) and to study its inhibitory effects on the invasiveness and migratory capacity of hepatocellular carcinoma (HCC) cell line HepG2 in vitro. METHODS: Total RNA was extracted from HCC. Then a 500 bp fragment at the 5' end of the human MMP2 cDNA sequence was synthesized by polymerase chain reaction (PCR) and was reversely inserted into the multiclone site (MCS) of the shuttle plasmid pAdTrack-CMV. With the resultant plasmid and the backbone plasmid pAdEasy-1, the homologous recombination took place in the E.coli BJ5183 and the recombinant adenoviral plasmid carrying the antisense MMP2 gene was constructed. The adenovirus (Ad-MMP2AS) was packaged and amplified in the HEK 293 cells and the viral titer was checked by GFP. Using the Boyden chamber model, the influence of Ad-MMP2AS on the invasion ability of HepG2 cells was determined in vitro. RESULTS: The recombinant adenovirus vector carrying antisense MMP2 was constructed successfully and a strong green fluorescence was observed in HepG2 cells under a fluorescence microscope. The viral titer was 1 x 10(8); Ad-MMP2AS can effectively inhibit the penetrating capacity of HepG2 cells through Matrigel in vitro. CONCLUSION: The recombinant adenovirus with antisense MMP2 can effectively inhibit the invasiveness and migratory capacity of HepG2 in vitro and may have potential in treating HCC.

[An experimental study of recombinant adenovirus microsphere carrying antisense multidrug resistance-associated protein gene for reversing MRP of hepatocellular carcinoma in vitro].

OBJECTIVE: To conduct an in vitro study on the effect of recombinant adenovirus microsphere encapusulated antisense MRP (as-mrp) for use in the gene therapy to overcome drug resistance in hepatocellular carcinoma. METHODS: Recombinant adenovirus microsphere encapusulated as-mrp was transfected into hepatocellular carcinoma multidrug resistance cells HepG2/ADM, the fluorescence intensity of transfected cells were observed at 48 hours and 120 hours after transfection. in vitro drug sensitivity was measured by MTT assay; the resistant index of andromycin resistant variants was determined by drawing the cell dosage reaction curves. The levels of MRP mRNA expression were detected by RT-PCR and the ratio of MRP mRNA/beta-actin was detected. Intracelluar rubidomycin (DNR) concertration was examined by flow cytometry (FCM). RESULTS: More than 90% of the HepG2/ADM cells could be transfected when microspheres being 10 mg. Adv microsphere inhibited the expression of mRNA in HepG2/ADM and enhanced the sensitivity of HepG2/ADM to chemotherapeutic drug. CONCLUSION: Recombinant adenovirus microsphere encapusulated as-mrp could effectively reverse HepG2/ADM cells, which would provide an experimental basis for the methods of reversing the multidrug resistance in human hepatocellular carcinoma.

[Construction of recombinant adeno-associated virus vectors carrying double gene of antisense multidrug resistance-associated protein and antisense multidrug resistance].

OBJECTIVE: To construct a recombinant adeno-associated virus vectors carrying double gene of antisense multidrug resistance-associated protein (MRP) and antisense multidrug resistance (MDR1) for use in studying the gene therapy to reverse the multidrug resistance (MDR) in hepatocellular carcinoma (HCC). METHODS: The 500 bp fragment (mrp) of MRP cDNA 5' region and the 600 bp fragment (mdr1) of MDR1 cDNA 5' region were amplified through polymerase chain reaction (PCR), and then they were linked to a combined gene fragment (mrp+mdrl) by overlapping technique. The combined gene fragment(mrp+mdrl) was cloned reversely into the multiple cloning site (MCS) of the expression plasmid pAAV-IRES-hrGFP in AAV Helper-Free System to construct the recombinant expression plasmid pAAV-IRES-hrGFP-(mrp + mdr1)AS. The packaging cell line (HEK 293 cell) was co-transfected with the pAAV-IRES-hrGFP-(mrp+mdr1)AS together with the control plasmid pAAV-RC and pHelper in AAV Helper-Free System by means of lipofectamine. The recombinant adeno-associated virus vector : rAAV2-(mrp+mdr1)AS carrying the double gene of antisense multidrug resistance-associated protein (MRP)and antisense multidrug resistance (MDR1) was packaged. Then the viral titer was checked by GFP. RESULTS: The recombinant adeno-associated virus vector : rAAV2-(mrp + mdr1)AS carrying antisense MRP and antisense MDR1 was constructed successfully, the strong green fluorescence was observed in HEK 293 cells under a fluorescence microscope. The viral titer was 2.5 X 10(6) efu/ml. CONCLUSION: The rAAV2-(mrp+mdr1)AS thus constructed could introduce the antisense MRP and antisense MDR1 into the human drug-resistant hepatocellular cell line effectively, which might provide a sound basis for the mechanisms and reversal methods of the multidrug resistance in HCC.

High level expression of bikunin in Pichia pastoris by fusion of human serum albumin.

Bikunin is a proteoglycan exhibiting broad-spectrum inhibitory activity against serine proteases and could potentially suppress tumor cell invasion and metastasis. Here, we have successfully expressed recombinant human bikunin (rh-bikunin) in Pichia pastoris and also established the purification procedure. Different fusion genes of h-UTI and domain I, domain I and domain II, domain I, domain II and domain III of human serum albumin (HSA) were inserted into expression vector pPICZαA. After expressed in shake flask, rh-bikunin was produced in an 30-L fermenter and purified by affinity chromatography and cation exchange chromatography. The final expression levels were 200 mg/L and we got totally 1.08 g (3650 IU/mg) of active purified rh-bikunin (purity is 98%) from 20 L of fermentation broth. The rh-bikunin consists of unique form with molecular masses of 25 kDa, and has the same N-terminals sequence as human native bikunin. This study provided a new method for high level expression of active rh-bikunin by using HSA as fusion parter.