Biomedical subjects

Ling Tian

Publications and source records attributed to Ling Tian.

At least 37 records · Page 2Linked to original sources

PubMed2005

[Expression of xenogeneic homologous epidermal growth factor receptor ectodomain in Pichia pastoris].

OBJECTIVE: To develop a secreted expression system of xenogeneic homologous epidermal growth factor receptor (EGFR) ectodomain using Pichia pastoris (P. pastoris), thus providing a basis for subsequent antitumor immunological researches on EGFR subunit protein vaccine. METHODS: In order to construct the secreted expression vector pPICZaA-mer and pPICZaA-her, we recombined the cDNA of xenogeneic (human) EGFR ectodomain (her) and the corresponding control mouse EGFR ectodomain (mer) with the empty plasmid pPICZaA, respectively. Then the recombinant plasmids were linearized by Sac I and transformed into P. pastoris strain GS115 by electroporation. The positive transformants were selected on YPDS plate including Zeocin and were induced to express by methanol. Consequently, the proteins in the culture supernatant were assayed with SDS-PAGE and Western-blot. So we could choose the high-level secreted expression recombinant strain to produce protein vaccine vastly. RESULTS: We got the secreted expression of mouse and human EGFR ectodomain in P. pastoris. The relative molecular mass of the two aimed proteins is about 95 x 10(3). The Western-blot analysis demonstrated that the expression proteins have much good antigenicity and specificity. CONCLUSION: The recombinant plasmids pPICZaA-mer and pPICZaA-her could be induced to express the EGFR aimed proteins by methanol in methanol-trophic yeast expression system.

Animals↗

PubMed2005

[Antitumor effects of recombinant quail vascular endothelial growth factor receptor 2, as a vaccine, combined with cisplatin on LL/2 tumor model in mice].

BACKGROUND & OBJECTIVE: Bio-chemotherapy is a new trend in cancer therapy. Antiangiogenic therapy represents a new strategy of tumor biotherapy. This study was designed to explore antitumor effect of recombinant quail vascular endothelial growth factor receptor 2 (qVEGFR) combined with cisplatin. METHODS: Lewis lung carcinoma model (LL/2) was established in C57BL/6 mice. Seven days after inoculation of tumor cells, mice were randomized into combination group, qVEGFR group, chemotherapy group, and normal saline (NS) group (10 mice/group), and received relevant treatments. Tumor growth, survival rate of mice, and side effects were observed. Anti-VEGFR-2 antibody-producing B cells (APBCs) was detected by enzyme-linked immunospot (ELISPOT), microvessel density (MVD) of tumor was detected by immunohistochemistry, and tumor cell apoptosis was also detected. RESULTS: Tumor volume of mice was obviously smaller in combination group than in NS group. Complete regression of tumor growth was observed in 3 of the 10 mice in combination group. Seventy days after inoculation of tumor cells, survival rate of mice was significantly higher in combination group than in qVEGFR, cisplatin, and NS groups (90% vs. 60%, 0%, and 0%, P < 0.05). APBCs counts were (156.8+/-19.3)/10(6) spleen cells in combination group, and (143.6+/-18.6)/10(6) spleen cells in qVEGFR group. MVD was significantly lower in combination group than in cisplatin, and NS groups (11.4+/-1.3 vs. 33.4+/-4.5, and 45.5 +/- 4.5, P < 0.01), MVD in qVEGFR group was 16.4+/-1.6. Tumor cell apoptosis was significantly higher in combination group than in qVEGFR, cisplatin, and NS groups [(36.2+/-3.5)% vs. (15.4+/-2.4)%, (17.6+/-2.6)%, and (4.1+/-1.4)%, P < 0.05]. CONCLUSION: The combination therapy of qVEGFR and cisplatin has synergistic antitumor effect.

Animals↗

PubMed2005

[Antitumor effects of interferon-gamma-inducible protein 10 combined with gemcitabine].

BACKGROUND & OBJECTIVE: Interferon-gamma-inducible protein 10 (IP-10), a member of alpha-chemoattractant family, is an inhibitor of tumor angiogenesis. Chemotherapy combined with antiangiogenic therapy has synergistic effects. This study was designed to explore antitumor effect of IP-10 combined with gemcitabine in tumor-bearing mice. METHODS: pBLAST-IP-10 was transferred into E.coli to prepare and purify plasmid IP-10. Hepatocarcinoma H22 model was established in BALB/c miceu Lewis lung cancer LL/2 model was established in C57BL/6 mice. The mice were divided into 4 groups, and treated with IP-10, gemcitabine, IP-10 plus gemcitabine, and normal saline (control), respectively. Protein level of IP-10 in serum was detected by ELISA. Survival rate of mice, tumor volume, and side effects of treatment were observed. Microvessel density (MVD) of tumor tissue was detected by immunohistochemistry. Apoptosis of tumor cells was detected by TUNEL method. RESULTS: After treatment of IP-10, IP-10 protein reached the peak level of (16.8+/-3.6) ng/ml at the 14th day, and remained a high level of (14.0+/-2.1) ng/ml at the 35th day. Compared with the controls, tumor volume of mice in the combination therapy group had significant regression, or disappeared. Nine weeks after inoculation of tumor cells, survival rate of mice was significantly higher in combination group than in IP-10, gemcitabine, and control groups (90% vs. 55%, 0%, and 0%, P < 0.005). No obvious side effects were observed. MVD of tumor tissues was significantly lower in combination group than in IP-10, gemcitabine, and control groups (15.8+/-2.4 vs. 45.6+/-2.0, 50.2+/-3.5, and 51.3+/-3.0, P < 0.01). At the 35th day after inoculation of tumor cells, apoptosis index was significantly higher in combination group than in IP-10, gemcitabine, and control groups (85.5+/-10.2 vs. 21.4+/-5.5, 8.4+/-2.0, 4.2+/-0.7, P < 0.01). CONCLUSION: Therapy of IP-10 combined with gemcitabine has significantly synergistic antitumor effect compared with IP-10 or gemcitabine alone.

Animals↗

PubMed2005

[Prokaryotic expression, purification and identification of recombinant Xenopus laevis and mouse vascular endothelial growth factors].

OBJECTIVE: To perform prokaryotic expression, purification and identification of Xenopus laevis and mouse vascular endothelial growth factors (VEGF). METHODS: The prokaryotic expression vectors were constructed by restriction endonuclease digestion and ligation, then the recombinant vector pET-xVEGF and the control vector pET-mVEGF were identified by restriction enzymatic digestion and DNA sequencing. The confirmed vectors were transformed into Escherichia coli. BL21 (DE3) and recombinant protein expression was induced by Isopropy-beta-D-thiogalactoside. The recombinant xVEGF and mVEGF were obtained and purified by Ni-NTA affinity chromatography under denature conditions and enterokinase specific digestion, respectively. Finally, the purified proteins' molecular weights and specificity were detected by SDS-PAGE and western blot analysis. RESULTS: Two new recombinant expression vectors, pET-xVEGF and pET-mVEGF, were constructed successfully. The xVEGF and mVEGF fusion proteins were expressed in E. coli. BL21 (DE3) stably, and the molecular weights of the purified xVEGF and mVEGF were identical to the expected values. The purity of final products reached a level higher than 95%. In addition, these two purified proteins could react with a specific antibody against mouse VEGF as expected. CONCLUSION: Recombinant Xenopus laevis VEGF and its control mouse VEGF protein may provide tools for further study of anti-tumor active immunity with this xenogeneic protein vaccine in mouse tumor models.

Animals↗

PubMed2005

[Fusion expression, purification and bioassay of IFN-gamma inducible protein-10 and thioredoxin gene in E. coli].

Interferon gamma-inducible protein 10, a member of the family of CXC chemokines, is secreted by interferon gamma-stimulated, monocytes, endothelial cells and keratinocytes. Interferon gamma-inducible protein 10 plays an important role in recruiting activated T cells into sites of tissue inflammation. In this experiment, PCR products of Interferon gamma-inducible protein 10 were cloned into prokaryote expression vector pET 32(a) to generate recombinant pET-IP10 with S-Tag at the N-terminus, and expressed successfully in E. coli BL21 (DE3). The total expressed products amounted to 25.3% in all bacterion proteins. pET-IP10 mainly formed inclusion body in E. coli. Soluble recombinant protein accounted for 20% among IP-10 fusion protein. The soluble recombinant proteins were purified by using S-Tag affinity chromatography effectively with purity of over 90%. The chemotaxis biological activity of purified Interferon gamma-inducible protein 10 could specifically exhibit the directional migration of stimulated T cells at concentration of 100 ng/ml. The results indicated that the strategy we used in this experiment was effective for recombinant Interferon gamma-inducible protein 10 production with biological activity.

Chemokine CXCL10↗

PubMed2005

[Nanobiotechnological gene transfer vectors in gene therapy].

Gene therapy has shown great potentials in the treatment of a number of human key diseases, such as genetic diseases, cancer and so on, yet there are still some big challenges to face. One of these challenges is how to develop efficient, safe, targeting and controlled release gene delivery systems. With the progress of nanobiotechnologies, some nanobiomaterials such as liposome, poly (D,L lactic-co-glycolic acid) and poly (L-lactide) have great potencies in developing the ideal gene transfer vector, just because of their properties of biocompatibility, bio-degradation, controllable release, easy modification for targeting and so on. In this review is presented a summary of the excellent properties of the nanobiotechnological gene delivery systems and their recent encouraging advances.

Biocompatible Materials↗

PubMed2005

[Effect of full-length cyclin B1 antisense cDNA on chemosensitivity of lewis lung carcinoma cells to gemicitabine in vitro and in vivo].

OBJECTIVE: To evaluate the effect of full-length cyclin B1 antisense cDNA (AS-CLB1) on chemosensitivity of Lewis lung carcinoma cells (LL/2) to gemicitabine (GEM) in vitro and in vivo and hence provide a therapeutic regiment for treating non-small cell lung (NSCL) cancer using AS-CLB1 combined with GEM. METHODS: Cell cycle phase distribution and apoptosis of LL/2 parent cells, LL/2/vect and LL/2/AS-CLB1 transfectants (LP, LV and LA cells) were determined by flow cytometry. In addition, the three kinds of cells were treated with GEM (20 nmol/L-20 micromol/L) in vitro for 1 h and 24 h respectively, and then cytotoxicity of GEM was measured by MTT assay. After inoculation with the three kinds of cells respectively, the C57BL/6 mice were treated with GEM [25 - 125 mg/(kg x day)] once every three days for four times when tumors developed; tumorigenicity and survival were observed and cell apoptosis in tumor tissues was determined by flow cytometry. RESULTS: LA cells displayed apparent apoptosis and G1 arrest compared with LP and LV cells (controls). Additionally, cytotoxicity of GEM to LA cells was more obvious than that to controls. Moreover, tumorigenicity was inhibited, cell apoptosis in tumor tissues was induced, and survival was evidently increased in LA cells group. CONCLUSION: AS-CLB1 slightly increased the sensitivity of LL/2 cells to GEM in vitro and in vivo. The function of AS-CLB1 may be associated with its ability to enhance the anti-tumor activity of GEM by inducing cell apoptosis and G1 arrest.

Animals↗

PubMed2005

[Antitumor effect of recombinant Xenopus laevis vascular endothelial growth factor (VEGF) as a vaccine combined with adriamycin on EL4 lymphoma in mice].

OBJECTIVE: To explore the antitumor effect of immunotherapy with recombinant Xenopus laevis vascular endothelial growth factor (xVEGF) as a vaccine combined with adriamycin on lymphoma model in mice. METHODS: EL4 lymphoma model was established in C57BL/6 mice. Mice were randomized into four groups: combination therapy, adriamycin alone, xVEGF alone and normal saline (NS) groups, and then were given relevant treatments. The growth of tumor, the survival rate of tumor-bearing mice, and the potential toxicity of regimens above were observed. Anti-VEGF antibody-producing B cells (APBCs) were detected by enzyme-linked immunospot (ELISPOT) assay. In addition, microvessel density (MVD) of tumor was detected by immunohistochemistry, and tumor cell apoptosis was also detected by TUNEL staining. RESULTS: The tumor volumes of mice were significantly smaller in combination group than those in other three groups (P < 0.05). Complete regression of tumor was observed in 3 of 10 mice in combination group. Forty-eight days after inoculation of tumor cells, the survival rate of mice was significantly higher in combination group than in NS group (P < 0.01). The anti-VEGF APBC count in combination group or xVEGF group was significantly higher, compared with that in adriamycin group or NS group (P < 0.01). MVD in tumor tissues was significantly lower in combination group than those in other three groups (P < 0.05). Moreover, tumor cell apoptosis was significantly higher in combination group than those in other three groups (P < 0.05). CONCLUSION: In this experimental study, the use of xVEGF vaccine and adriamycin as a combination of immunotherapy with chemotherapy has sucessfully produced synergistic antitumor effect on lymphoma in mice.

Animals↗

PubMed2005

[Study on the expression of HSP70 and HSP90beta in nasopharyngeal carcinoma and the clinical significance].

OBJECTIVE: To study the expression of HSP70 and HSP90beta and its clinical significance in human nasopharyngeal carcinoma. METHOD: Immunohistochemical method SP was used to detect the expression of HSP70 and HSP90beta in 50 cases and its clinical significance were studied. RESULT: The positive rate of HSP70 and HSP90beta were 72% and 56% respectively. Analysis of patients, survival demonstrated that the prognosis of NPC with HSP70 (-) and HSP90beta (-) expression were significantly better than the others. CONCLUSION: The overexpression of HSP70 and HSP90beta were probably concerned with the occurrence, development and prognosis of nasopharyngeal carcinoma.

Adolescent↗

PubMed2005

[Screening and identification of human nasopharyngeal carcinoma-associated antigens].

OBJECTIVE: The purpose of this study was to provide an informative marker for clinical immunopathological diagnosis of nasopharyngeal carcinoma (NPC) and to lay a foundation for furture study of immunotherapy. METHODS: The NPC cDNA expression library was screened by serological analysis of recombinant cDNA expression libraries (SEREX) technology using xenogenic immune sera. The positive nucleotide sequences of cDNA inserts were determined and analysed with DNASIS and BLAST software in EMBL and GenBank. RESULTS: Eighteen positive clones encoding antigen genes were obtained after immunoscreening. These antigen genes included known genes, such as MAGE gene, CREM gene, beta2-microglobulin, Ribosomal Protein, et al, and unknown genes or ESTs in GenBank. To characterize the expression profile of these genes, the antibodies in sera from 40 nasopharyngeal carcinoma patients and 40 healthy donors were assayed with three antigens to screen specific and relative serum markers for nasopharyngeal carcinoma. The results showed that the positive rates for These results indicate nasopharyngeal carcinoma patients were higher than those for healthy donors. CONCLUSION: that some of the antigens may be related to nasopharyngeal carcinoma and may be valuable tumor markers in the diagnosis of nasopharyngeal carcinoma.

Antigens, Neoplasm↗

PubMed2005

[Construction of the recombinant adenovirus vector expressing human CD40 ligand gene].

OBJECTIVE: To construct a recombinant adenovirus vector expressing hCD40L gene and explore it in the use of anti-tumor gene therapy. METHODS: 1,900 bp gene fragment was obtained form plasmid pORF-hCD40L by Xho I/Swa I cutting and then cloned directionally into the pShuttle plasmid, finally, the resultant plasmid was digested by restriction endonnuclease PmeI and subsequently cotransformtion into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant and then the recombinant was packaged in the 293 cells. Some methods such as PCR and endonulease digestion were employed to identify the recombinant adenovirus. RESULTS: The evidences of endonulease digestion and PCR analysis confirmed that recombinant hCD40L gene was correctly inserted into adenovirus vector. CONCLUSION: The adenoviral vector which expressed hCD40L gene was constructed. It provides an experimental basis for studies on it expression in the mammalian cells and in tumor gene therapy.

Adenoviridae↗

PubMed2004

Combination of low-dose cisplatin and recombinant xenogeneic endoglin as a vaccine induces synergistic antitumor activities.

Angiogenesis is critical to the growth and metastasis of solid tumors, and acquired drug resistance is one of the major hindrances to chemotherapy. Thus, we sought a rational strategy using the combination of antiangiogenic biotherapy and chemotherapy for cancer therapy. We explored the efficacy of a strategy combining low-dose cisplatin and a recombinant xenogeneic endoglin as a protein vaccine, which we previously demonstrated to have effective antiangiogenic effects in several mouse models. We found that both low-dosage cisplatin and xenogeneic endoglin vaccine individually resulted in effective suppression of tumor growth in 2 tumor models via inhibition of tumor angiogenesis. Remarkably, the combination therapy resulted in not only significant antiangiogenic effects but also additional promotion of tumor cell apoptosis and inhibition of tumor cell proliferation, without any ensuing increase in host toxicity during the course of treatment, which lasted for 6 months. In addition, the combination demonstrated a synergistic relationship, which was shown in all of the synergistic indexes, i.e., tumor volume, angiogenesis, apoptosis and proliferation. Both antibodies and antibody-producing B cells against mouse self-endoglin were observed in all mice immunized by the xenogeneic endoglin vaccine (alone and combination), which suggested that low-dose cisplatin did not suppress the host immune response but potentiated the antitumor activity of the xenogeneic endoglin vaccine. These observations may provide the basis for an effective alternative strategy for cancer therapy in the near future.

Animals↗

PubMed2004

Induction of apoptosis and tumor regression by vesicular stomatitis virus in the presence of gemcitabine in lung cancer.

Vesicular stomatitis virus (VSV) has been shown to replicate rapidly in vitro and kill selectively a variety of tumor cell lines. The present study was designed to determine whether gemcitabine potentiates the antitumor activity of VSV in vitro and in vivo. A549 human lung adenocarcinoma cells and LLC Lewis lung carcinoma cells were treated with VSV (0.1-10 plaque-forming units per cell) plus gemcitabine (20 nM to 20 microM). Mice bearing A549 or LLC were treated with VSV (5 x 10(4) to 1 x 10(8) plaque-forming units) daily for 5 days plus gemcitabine (5-125 mg/kg/day) once every 3 days for 4 times. Induction of apoptosis and effects on growth inhibition were assessed. The lung cancer cells treated with VSV plus gemcitabine displayed the apparently increased apoptotic cells compared with treatment with VSV or gemcitabine alone. The combined treatment with VSV plus gemcitabine induced the apparent antitumor activity with complete regression of the established lung cancer in both A549 and LLC lung cancer models and augmented the induction of apoptosis in lung cancer cells in vivo as well. This study suggests that the combined treatment with VSV plus gemcitabine may augment the induction of apoptosis in lung cancer cells in vitro and in vivo, and that the augmented antitumor activity in vivo may result from the increased induction of apoptosis in lung cancer cells. The present findings may be of importance to the further exploration of the potential application of this combined approach in the treatment of lung cancer.

Adenocarcinoma↗

PubMed2004

Active immunotherapy of tumors with a recombinant xenogeneic endoglin as a model antigen.

Angiogenesis play a critical role in tumor growth and metastasis. Increasing evidence suggests that endoglin is a powerful marker of angiogenesis in solid malignancies. Thus, breaking of immune tolerance of self-endoglin-associated angiogenesis is an attractive approach to cancer therapy. To test this concept, we recombined the extracellular domains of porcine endoglin, and used it as a xenogeneic vaccine. We found that immunotherapy with porcine endoglin was effective at both protective and therapeutic anti-tumor immunity in several mouse tumor models. Autoantibodies against mouse endoglin were identified by Western blot and ELISA. IgG1 and IgG2b were substantially increased. Anti-endoglin antibody-producing B cells were detectable by ELISPOT assay. There was endothelial deposition of immunoglobulins within tumors. The anti-tumor activity was also induced by the adoptive transfer of the purified immunoglobulins. Angiogenesis was apparently inhibited within the tumor tissues and on the alginate beads. The increased apoptotic cells were found within the tumor tissues from the mice treated with porcine endoglin. The anti-tumor activity and production of autoantibodies against mouse endoglin could be abrogated by depletion of CD4(+) T lymphocytes. Remarkably, no marked toxicity was found in the immunized mice. These observations may provide an alternative rational strategy for active cancer immunotherapy.

Adoptive Transfer↗

PubMed2004

Immunotherapy of tumor by targeting angiogenesis.

Tumor growth and metastasis are angiogenesis-dependent. Anti-angiogenic therapy represents a new strategy for the development of anti-cancer therapies. In recent years, there has been made great progress in anti-angiogenic therapy. As far as the passive immunotherapy is concerned, a recombinant humanized antibody to vascular endothelial growth factor (VEGF)--Avastin has been approved by FDA as the first angiogenesis inhibitor to treat colorectal cancer. For active specific immunotherapy, various strategies for cancer vaccines, including whole endothelial cell vaccines, dendritic cell vaccines, DNA vaccines, and peptides or protein vaccines, have been developed to break immune tolerance against important molecules associated with tumor angiogenesis and induce angiogenesis-specific immune responses. This article reviews the angiogenesis-targeted immunotherapy of tumor from the above two aspects.

Antibodies, Monoclonal↗

PubMed2004

[Experimental studies on anti-tumor effects of BLC-modified tumor cell vaccine combined with cisplatin].

BACKGROUND & OBJECTIVE: Chemokines are small molecule proteins that are the main mediators of cell migration. Studies indicated that chemokines could induce antitumor immune response strongly and steadily. At present, there was not antitumor therapy about chemokine BLC combined with chemotherapy. This study was designed to explore the anti-tumor effects and mechanisms of BLC-modified tumor cell vaccine combined with cisplatin. METHODS: Firstly, we transfected pcDNA-BLC into murine tumor cell lines and established stable transfected tumor cell lines (Colon26 and LL/2) to express BLC. Null plasmid pcDNA3.1 (+) was also transfected into Colon 26 or LL/2 cells stably for comparison. We established mouse model with pcDNA-BLC stable transfected tumor cells (Tc), control mouse model with pcDNA3.1(+) stable transfected tumor cells (Pc), and mouse model with parental tumor cells (Nc). Then the 60 mice were inoculated subcutaneously (s.c.) in the right flanks with a total of 5x10(5) viable tumor cells (Tc to 20 mice, Pc to 20 mice, and Nc to 20 mice, respectively; 6-8 week BALB/c female mouse in colon 26 mouse model or 6-8 week C57BL/6 female mouse in LL/2 mouse model). The groups (Tc, Pc, and Nc) were randomly subdivided into Group A and Group B with 10 mice for each, resulting in Tc-A/Tc-B, Pc-A/Pc-B, and Nc-A/Nc-B, respectively. Group A (including Tc-A, Pc-A, and Nc-A) as chemotherapy group was injected (i.p.) with 0.1 ml cisplatin at the concentration of 2 mg/kg once a week for two weeks; Group B (including Tc-B, Pc-B, and Nc-B) as control with 0.1 normal saline. We further observed anti-tumor activity including the tumor growth, mice survival rate, side effects as well as tumor morphological analysis. Apoptotic cells were also determined in tumor tissues. RESULTS: The combination therapy group Tc-A showed significant anticancer activities. The tumor growth was inhibited efficiently with 3/10 mice showing complete regression in LL/2 mouse model and 1/10 mice showing complete regression in Colon 26 mouse model. The combination therapy group Tc-A showed that the survival rate of mice was 100% in the two mouse models, compared with 68% in group Tc-B in C57BL/6 mouse model and 65% in group Tc-B in Colon 26 mouse model and those in other groups. In addition, the combination therapy group Tc-A induced tumor cell apoptosis significantly. CONCLUSIONS: The therapy of BLC-modified tumor cell vaccine combined with cisplatin had significant synergistic effect against tumor. It might develop a new approach for specific immunotherapy of tumors.

Animals↗

PubMed2004

[Prokaryotic expression, purification and refolding of extracellular ligand binding domains of chick Tie-2 and its immunogenicity].

OBJECTIVE: To study the prokaryotic expression of extracellular ligand binding domains of chick Tie-2, the purification and refolding conditions of the recombinant protein, and to observe its immunogenicity in mouse. METHODS: A DNA fragment encoding extracellular ligand binding domains of chick Tie-2 was obtained by PCR from a previous constructed plasmid as a template. The amplified fragment was then inserted into prokaryotic expression vector PQE30, and was expressed in E. coli XL-1 blue by adding isopropyl-beta-D-thiogalactoside(IPTG). The recombinant protein in inclusion bodies was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography under denatured conditions. Then the refolding of the purified protein was performed with gradient dialysis. The target protein was injected into mouse subcutaneously, and the antiserum of the mouse was analyzed by ELISA and Western blot analysis. RESULTS: The recombinant protein was highly expressed in E. coli XL-1 blue, and in mouse it produced the antibody which could specifically recognize the recombinant protein. CONCLUSION: The protein of extracellular ligand binding domains of chick Tie-2 can be highly expressed in prokaryotic expression system, and the expressed protein can induce immune response in mouse. These findings are very important for the further study of this protein in anti-angiogenesis and immunotherapy research.

Animals↗

PubMed2004

[Prokaryotic expression of extracellular ligand binding domains of chick tie-2 and its anti-angiogenesis effect].

OBJECTIVE: To study the prokaryotic expression of extracellular ligand binding domains of chick tie-2, the purification, refolding conditions of the recombinant protein, and its anti-angiogeneic effect. METHODS: A DNA fragment encoding extracellular ligand binding domains of chick tie-2 was obtained by PCR amplification using a previous constructed plasmid as a template. The amplified fragment was then inserted into prokaryotic expression vector pQE30, and was expressed in E.Coli XL-1 blue by adding isopropyl-beta-D-thiogalactoside(IPTG). The recombinant protein in inclusion bodies was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography under denatured conditions. Then the refolding of the purified protein was performed with gradient dialysis. The target protein was injected s.c. into mouse, and the antibody was detected by ELISA and Western blot analysis. The antibody was purified from the antiserum and then incubated with human umbilical endothelial vein cell (HUEVC) to find its anti-angiogenesis in vitro by using propidium iodide(PI) dying through FACS. Alginate encapsulated tumor cell assays were performed and micro-vessel density was determined by counting per high power field in the sections stained with an antibody reactive to CD31 to test its inhibition of angiogenesis. RESULTS: The recombinant protein was highly expressed in E.Coli XL-1 blue, and the antibody produced in mouse could specifically recognize the recombinant protein. The purified antibody could induce apoptosis of HUEVC in vitro. The anti-angiogenic effect of the antibody could also be found in alginate-encapsulate tumor cell assay and by counting micro-vessel density. CONCLUSION: The protein of extracellular ligand binding domains of chick tie-2 can be expressed at high level in the prokaryotic expression system, and the expressed protein can induce immune response in mouse. Furthermore, the antibody can induce the anti-angiogenic effect.

Angiogenesis Inhibitors↗