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A set of backcross population were constructed with Suinong 14 as recurrent parent and wild soybean ZYD00006 as donor parent. 16 QTLs (14 QTLs with positive effect, 2 QTLs with negative effect) for protein content among 9 linkage groups detected by composite interval mapping (CIM) with Win QTL2. 5. Ten BC3F3 lines with high protein content compared with Suinong 14 were screened out, and another 10 lines from BC3F3 were used as control (random population) . 17 QTLs for protein content on 10 linkage groups were detected with genotype analysis by chi - square on genetic hitchhiking, and all showed positive effect. 7 QTLs were detected by both methods. These materials and markers provided important material foundation and marker information for gene cloning and molecular assisted breeding in high protein content. [ABSTRACT FROM AUTHOR]

A comparative study on the reactivity of Shenmu char with CO2 and steam was conducted at the temperatures from 900 °C to 1 050 °C using isothermal thermogravimetric analysis method. To better understand the reaction activity of residual carbon, the homogeneous model, the shrinking un-reacted core model, the integrated model and the modification volumetric model were applied to correlate the relationship between reaction rate and carbon conversion at the late gasification stage. The results show that both the integrated model and the modification volumetric model have a better fitting accuracy. The fitting accuracies of the homogeneous model and the shrinking un-reacted core model change with the gasification temperatures and the agents. At the late stage, the ranges of the apparent activation energy of Shenmu char in the atmosphere of CO2 and steam are 200. 65-231.00 kJ/mol and 105.48-169. 10 k J/tool, respectively. The apparent activation energy calculated with the modification volumetric model is approximately 36 kJ/mol higher at the late stage than that at the early and middle stages. Shenmu coal char gasification was under chemical reaction control at the late stage within the experimental temperature range. [ABSTRACT FROM AUTHOR]

Based on the staged conversion technique of brown coal pyrolysis, partial gasification and residual carbon combustion in circulating fluidized beds, rapid and slow coal char were produced from the brown coal of Shigouyi coal mine in Ningxia, at the temperatures from 700 °C to 950 °C in a horizontal tube furnace. The effects of pyrolysis conditions on the surface morphology and structure of the coal chars were investigated. A contrastive study was conducted using thermogravimetric technology, testing the reactivity of coal chars gasification with steam and CO2. The effects that gasification temperature, pyrolysis temperature and pyrolysis rate have on the gasification reactivity of coal chars were also analyzed. The results show that gasification temperature is the most important factor influencing gasification rate, while pyrolysis temperature has a weaker effect. However, there is great difference in gasification reactivity between steam and CO2, which decreases as gasification temperature rises. Compared to steam gasification, pyrolysis conditions have greater influence on CO2 gasification. The relation of reaction indexes between steam and CO2 gasification can be expressed using a quadratic regression equation, and a linear relationship exists between the distributed activation energy of both reactions in a carbon conversion range from 10% to 80%. [ABSTRACT FROM AUTHOR]

In this study, the hitchhiking effects in a backcross population with strong selection under salty and drought conditions were analyzed by scanning multiple molecular loci, and 34. 38% of the loci were found with segregation distortion in random population. After drought and salt tolerance selection, the segregation distortion ratio increased to 51. 56% and 55. 88%. SSR alleles showed a serious distortion and genotype structure in backcross introgression lines changed considerably after drought or salt tolerance selection. Three putative drought - tolerance related loci (Satt338 , Satt640 and Sat_108) and five putative salt - tolerance loci (Sat_271, Satt726, Satt640, Satt513 and Sat_108) were obtained with chi - square test (P < 0.000 1) based on hitchhiking effect calculated by comparison of the introgressive frequency of alleles between random population and selected population. [ABSTRACT FROM AUTHOR]

To evaluate the efficacy and safety of interferon-alpha-2b (IFN-α-2b) in polycythemia vera patients(PV patient) with or without post-polycythemic myelofibrosis (post-PV MF), 30 patients with mutated JAK2V617F were enrolled in this study, from which 29 patients were evaluable. The percentage of mutated JAK2V617F allele (V617F%) was evaluated by real-time polymerase chain reaction (RT-PCR) before and after treatment with IFN-α-2b. The correlation of V617F allele burden with the major clinical outcomes was studied. Adverse effects appeared in patients was observed. The results showed that the median follow-up was 24 (12 - 42) months for 29 evaluable patients. Complete hematologic response was achieved in 10%, 48%, 72% and 78% of patients after treatment for 6, 12, 24 and 36 months respectively. The detection of V617F allele burden revealed that the molecular remission of patients (V617F%) was achieved in 41%, 76%, 89% and 89% after treatment for 6, 12, 24 and 36 months respectively. Molecular complete remission (JAK2V617F undetectable) was achieved in 4 patients, lasted from 6 to 12 months after IFN-α-2b discontinuation. The decrease of V617F% in patients with post-PV MF was significantly higher than that in patients without post-PV MF (53 ± 18% vs 32 ± 22%, respectively; p = 0.031) after treatment for 12 months. PV patients had a good tolerance to IFN-α-2b. It is concluded that IFN-α-2b can decrease the mutated V617F allele burden. Patients with PV, especially with post-PV MF, can achieve molecular remission after treatment with IFN-α-2b.

Objective: To detect the expression of the protein of TGF-beta1 and E-cadherin in the primary and metastatic lesions of ovarian carcinoma and explore the mechanism of the metastasis of ovarian carcinoma., Methods: Immunohistochemistry (IHC) was performed to detect the expression of TGF-beta1 and E-cadherin proteins in primary and metastatic ovarian carcinoma, benign epithelial ovarian tumor and normal ovarian tissue., Results: The expression of TGF-beta1 was significantly higher in ovarian carcinoma (67.2%) than in benign tumors (28.6%) and normal ovarian tissue (18.9%) (Chi2=26.94, P<0.001), but E-cadherin expression showed a reverse pattern. TGF-beta1 expression in the primary ovarian carcinoma carcinoma was associated with the FIGO stage, lymph metastasis and ascites of the tumor (P=0.01, P=0.01, and P=0.04, respectively). E-cadherin expression in the tumor was associated with the differentiation (P=0.02) and lymph metastasis of ovarian carcinoma (P=0.04). The expressions of TGF-beta1 and E-cadherin were all significantly lower in the primary tumors than in the metastatic tumor (Chi2=4.70, P=0.03; Chi2=5.91, P=0.015). A significant correlation was found between the expressions of the TGF-beta1 and E-cadherin in the primary carcinoma (Kappa value of -0.32, P=0.01)., Conclusion: TGF-beta1 and E-cadherin are closely associated with the metastasis of ovarian carcinoma and might be potential targets for controlling the metastasis of ovarian carcinoma.

Objective: To investigate the role of Ara-C in regulating anti-CD3/anti-Pgp mediating T-lymphocytes activities against multi-drug resistant leukemia cells., Methods: The diabody of anti-CD3/anti-Pgp was purified by E-tag affinity chromatography. K562 and K562/A02 cells were treated with Ara-C. The expressions of B7-1 and B7-2 on K562 and K562/AO2 cells were detected by FACS. The cytotoxicity of T-lymphocytes combined with anti-CD3/anU-Pgp plus Ara-C was analyzed by CytoTox 96 nonradioactive method., Results: The expressions of B7-1 and B7-2 on K562 and K562/A02 cells treated by Ara-C was significantly higher than those untreated. The effect/target ratio was from 0.39:1 to 25:1, and the killing rate of activated T cells to anti-drug-resistant leukemia cells was from (16.44 +/- 1.20)% to (60.49 +/- 2.90)%. The killing rates were increased gradually, with both the effect/target ratio and the antibody concentration increasing (P < 0.05)., Conclusion: Ara-C may be an important adjuvant for improving anti-CD3/anti-Pgp mediating T-lymphocytes activities against multi-drug resistant leukemia cells.

Objective: To investigate the effect of calmodulin antagonist O-(4-ethoxyl-butyl)-berbamine (EBB) on proliferation of human breast cancer cell MCF-7 and its possible mechanism., Methods: MTT assay was used to analyze the effect of EBB on tumor cells growth. Flow cytometry was used to detect its impact on the cell cycle of MCF-7 cells. Immunofluoresce labeling technique and laser scanning confocal microscope were used to reveal the changes of the microtubule, microfilament, mitochondrion, and endoplasmic reticulum in the cells., Results: The IC50 value of EBB in MCF-7 cells was (13.0 +/- 3.7) micromol/L. MCF-7 cells were arrested at S phase after EBB treatment. Meanwhile, depolymerization of the microtubule and microfilament, impairment of the mitochondrion and swelling of endoplasmic reticulum were observed., Conclusion: EBB arrests MCF-7 cells at S phase by inhibiting the growth of MCF-7 cells, which may be related to the changes of structures and functions of the microtubule, microfilament, mitochondrion, and endoplasmic reticulum.

Objective: To establish a three-step purification method of preparative-scale antiCD20 (Fab')2 using AKTA prime., Methods: AntiCD20 (Fab')2 was extracted by hyperosmotic solution and then purified by CM sepharose FF, phenyl sepharose FF, and protein G sepharose FF., Results: Around 8 mg anti-CD20 (Fab')2, whose purification was 96.678%, was purified. The antigen-binding activity of antiCD20 (Fab')2 was similar to that of antiCD20 (Fab')2 purified by protein G sepharose FF and S-100., Conclusion: The three-step purification method can obtain high-purity preparative-scale antiCD20 (Fab')2 in a simple way.

Objective: To study the synergistic mechanism between PH II -7 and doxorubicin against multi-drug resistant HL-60/ADR cells and its parent HL-60 cells., Methods: The anti-tumor activity of doxorubicin alone and combined with PH II -7 were measured by MTT assay. RNA was extracted from the cells treated with PH II -7 for different times or doses then the expression of MRP gene was measured by RT-PCR. Confocal laser scanning microscopy and FACS were used to detect the intracellular cumulation of doxorubicin in PH II -7 treated HL-60 and HL-60/ADR cells., Results: PH II -7 has anti-tumor effect with IC50 of (0.83 +/- 0.08) micromol/L and (1.74 +/- 0.56) micromol/L for HL-60 and HL-60/ADR, respectively. It could potentiate the anti-tumor effect of doxorubicin with CDI of 0.7 and 0.43 for HL-60 and HL-60/ADR, respectively. PH II -7 and doxorubicin act synergistically in inhibiting the proliferation of HL-60 and HL-60/ADR cells and down-regulating the expression of MRP gene in a dose and time dependent manner. PH II -7 restored the intracellular cumulation of doxorubicin in HL-60/ADR cells to 55% of that in HL-60 cells., Conclusion: PH II -7 can significantly hasten the cytotoxicity of doxorubicin to HL-60 and HL-60/ADR cells through down-regulating the expression of MRP. The synergistic effect was more obvious in HL-60/ADR cells.

Objective: To prepare monoclonal antibody (McAb) against anti-CD3 ScFv for purifying and detecting serum anti-CD3 antibody concentration., Methods: McAb against anti-CD3 ScFv was prepared by hybridoma technique and used to prepare affinity chromatography column, which was used to purify anti-CD3 ScFv and Diabody [CD3 x Pgp] without E-tag. The binding activities of anti-CD3 ScFv, Diabody [CD3 x Pgp] without E-tag, and Diabody [CD3 x Pgp] purified by anti-CD3 affinity chromatography column or anti-E-tag affinity chromatography column against K562/A02 cell and Jurket cells were detected by fluorescence activated cell sorting (FACS) method. ELISA was used to identify the specificity of the McAb., Results: McAb against anti-CD3 ScFv specifically detected serum anti-CD3 ScFv without reacting with sera. The anti-CD3 ScFv purified by anti-CD3 affinity chromatography column and purified by anti-E-tag affinity chromatography column had the same specific binding activity with Jurkat cells. The positive binding rates of Diabody [CD3 x Pgp] without E-tag to K562/A02 and Jurkat cells were 89.87% and 83.95%, respectively. In the competitive binding experiments with K562/A02 and Jurkat cells, the binding rates of Diabody [CD3 x Pgp] without E-tag decreased to 56.30% and 43.78%, respectively., Conclusion: The McAb against anti-CD3 ScFv prepared in our lab can be used to purify and detect serum anti-CD3 antibody concentration.

Aim: To observe the effects of Ara-c on the expression of CD86 molecule on acute leukemia cells and explore the possible mechanisms., Methods: The expression of CD86 on U937, HL-60 and NB4 cell lines treated with or without Ara-C wa assayed by flow cytometry. The mRNA expression of CD86, NF-kappaB, IFN-gamma was examined by semiquantitative RT-PCR., Results: UP-regulation of CD86 was observed on those cells treated by Ara-c. The level of CD86 and NF-kappaB mRNA in Ara-c treated cells was significantly enhanced. IFN-gamma was detectable 72 hours after T cell activation., Conclusion: Ara-C can enhance CD86 and NF-kappaB expression on acute leukemia cells, which may play critical role in T cell activation and differentiation.

Aim: To construct and express human 4-1BBL/anti-CD20 bispecific fusion protein and identify its biological activity., Methods: PCR and overlapping PCR were used to construct human 4-1BBL/anti-CD20 bispecific fusion protein. DNA sequencing was performed by the terminus of the fusion protein nucleotide.The product was purified by affinity chromatography and analyzed by Western blot and its antigen-binding activity was examined by FACS., Results: The data of DNA sequence showed that human 4-1BBL/anti-CD20 bispecific fusion protein was correct. The fusion protein was recovered in high yield (up to 4 mg/L) after E-tag purification and predominantly(90%) as a dimer. The fusion protein could bind to Raji cells(CD20(+)) and A549 cells(4-1BB(+)), respectively., Conclusion: The human 4-1BBL/anti-CD20 bispecific fusion protein with high level expression was successfully obtained and could bind to Raji ceIls and A549 cells.

Aim: To investigate the role of the extracellular domain of human 4-1BBL (ex4-1BBL) in regulating the in vitro activities of peripheral blood lymphocytes (PBL)., Methods: Viable cells were quantified with Trypan-blue exclusion assay. CytoTox 96 Nonradioactive Cytotoxicity Assay Kit was used for measuring LDH levels of supernatant. ELISA kit was used for measuring IL-2 level. In vitro cytotoxity of PBL combined with anti-CD3/anti-Pgp bispecific diabody plus ex4-1BBL was analyzed with CytoTox 96 nonradioactive method., Results: Ex4-1BBL can increase the proliferation of PBL, reduce cell death, promote IL-2 secretion, and the experimental group with ex4-1BBL showed obviously enhanced cytotoxic effect toward K562/A02 cells., Conclusion: Ex4-1BBL may be an important adjuvant for improving activities of PBL.

Aim: To construct and express a diabody [CD3 x Pgp] without Etag and analyse its biological activity., Methods: In this study, the diabody [CD3 x Pgp] was obtained by PCR and restriction cleavage, and expressed in E.coli 16C9. The product was purified by anti-anti-CD3 scFv affinity chromatography and verified through SDS-PAGE electrophoresis. Flow cytometry(FCM) was used to analyse the bingding properties and competitive bingding capacity., Results: The sequence of diabody [CD3 x Pgp] without Etag was correct. It migrated as two bands with the expected molecular weight(25 kD and 26 kD) in SDS-PAGE. The binding rate to CD3 and Pgp antigen was 83.95% and 89.87% respectively. The competitive bingding rate to CD3 and Pgp was 43.78% and 50.25% respectively., Conclusion: The diabody [CD3 x Pgp] without Etag has been successfully constructed, expressed and purified. The product can bind to CD3 and Pgp antigen specifically, and its biological activity doesn't decrease.

Objective: To study the specific cytotoxicity mediated by anti-P-gp/anti-CD(3) diabodies in multidrug resistant solid tumor using P-gp as target., Methods: The anti-P-gp/anti-CD(3) diabodies were secreted from E. coli strain 16C9 containing the expression plasmid PAYZDCP, grown at 30 degrees C in a shaker flask; the diabodies were purified by affinity chromatography and identified by SDS-PAGE; the effect of the anti-P-gp/anti-CD(3) diabody mediated lysis of P-gp-expressing tumor cells was assayed by (51)Cr release assay in vitro, and by human KB nude mouse xenograft models in vivo., Results: The diabodies were generated by bacteria as a soluble functional form and purified by one-step affinity chromatography with a yield > 4 mg/L culture medium. In (51)Cr release assay, the diabodies targeted human activated T cells to lyse P-gp(+)-KB/MDR cells in a dose-dependent manner. It suggested that the diabody was able to induce an efficient lysis of the target cells by human T cells in vitro. When combined with activated human T cells, the diabody significantly inhibited the growth of KB/MDR, but had no effect on KB xenografts., Conclusion: The anti-P-gp/anti-CD(3) bispecific antibody is a potent agent for targeting human T lymphocytes to lyse solid tumor cells overexpressing P-gp in vitro and in vivo.

Objective: To study the specific targeting cytotoxicity to drug-resistant leukemia cells mediated by anti-Pgp/anti-CD3 diabody., Methods: The diabody was purified by affinity chromatography and identified by SDS-PAGE and FACS. The effect of the anti-Pgp/anti-CD3 diabody mediated lysis of Pgp-expressing tumor cells was assayed by human leukemia nude mice xenograft model in vivo., Results: The diabody was produced in E.coli in a soluble functional form and could bind both Jurkat cells (CD3(+)) and K562/A02 cells (Pgp(+)). The binding rates were 86.25% and 86.26%, respectively. It could inhibit tumor growth by 98.57% and prolonged the survival of mice bearing xenografted K562/A02 cells., Conclusion: The diabody was proved to be a potent agent for mediating T lymphocyte cytotoxicity to lyse Pgp expressing tumor cells in vitro and in vivo.

Aim: To construct and express a single chain antibody (scFv) against human vascular endothelial growth factor (VEGF) receptor KDR and characterize its biological activity., Methods: The restriction enzyme sites were added to the previously cloned V(H) and V(L) genes of mAb Ycom1D3 against KDR by PCR. The anti-KDR scFv gene was constructed by the splicing overlap extensive (SOE) PCR and then inserted into fusion expression vector pAYZH. The recombinant protein was expressed in E.coli 16C9 and purified with His-tag affinity chromatography. The specificity of the purified scFv was examined by ELISA and FACS., Results: DNA sequencing indicated that the cloned scFv gene consisted of 729 bp, encoding 243 amino acids. After induction in low-phosphate medium of AP5, a new protein band with relative molecular mass (M(r)) of 30 000 appeared on gel of SDS-PAGE and on nitrocellulose membrane of Western blot, which was consistent with the theoretically predicted value. Anti-KDR scFv was expressed in the form of inclusion body, which accounted for 20% of total bacterial protein. ELISA and competitive immunofluorescence binding test showed that the anti-KDR scFv had the same binding activity as mAb Ycom1D3 and that it could block VEGF/KDR interaction effectively., Conclusion: Recombinant anti-KDR scFv gene has been successfully constructed and expressed in E.coli 16C9, which lays the foundation for its diagnostic and therapeutic application.

Background & Objective: The anti-CD20 antibodies and fragments have been applied for treatment of non-Hodgkin's lymphomas (NHL) in clinic. The new anti-CD20 antibodies and their fragments (unmodified or radiolabeled) yet have been exploited for those patients with incomplete response to rituximab. The chimeric antibody fragments Fab and F(ab) '(2) derived from HI(47)(a mouse anti-CD20 antibody) have been constructed. The present study was designed to determine the effect of HI(47) and chimeric anti-CD20 antibody fragments on growth inhibition and apoptosis of lymphoma cells., Methods: The binding of anti-CD20 antibodies to CD20 positive human B cell lymphoma Raji cells was examined using immunofluorescence assay. MTT method was used to evaluate the effect of chimeric antibody fragments on Raji cells growth. Annexin V staining and DNA ladder were used to examine the apoptosis of Raji cells induced by chimeric antibody fragments., Results: HI(47) and its chimeric antibody fragments were capable of binding to CD20 positive Raji cells with the binding rate of above 90%. HI(47) did not compete with rituximab in binding with Raji cells. The growth of Raji cells was inhibited by HI(47) and its fragments with the inhibition rates of (57+/-1.5)%, (65.2+/-2.5)%,and (77.2+/-3.2)%, respectively, at the concentration of 100 microg/ml. The monovalent antibody fragment Fab (20 microg/ml) induced the apoptosis of Raji cells with early apoptosis rate of 17%., Conclusion: The chimeric anti-CD20 antibody fragments derived from HI(47) have inhibitory effect on Raji cells and can induce apoptosis of Raji cells.

Monoclonal antibodies (mAb) directed against CD20, either unmodified or in radiolabeled forms, have been successfully exploited in clinic as effective therapeutic agents in the management of non-Hodgkin's B-cell lymphoma. The antibody fragment is a potential agent in image and therapy of tumor. To further improve the soluble expression of anti-CD20 antibody Fab' fragment, PCR was used to mutate the anti-CD20 VL and VH genes and its biological activity was identified. The expression vector of chimeric antibody Fab' was constructed and expressed in E. coli. The data of mutant clone DNA sequence showed that the amino acid of light chain gene of the parent anti-CD20 antibody (H47) was successful mutated as Ser (GAG)-Asn (CAG). The soluble expression of mutated anti-CD20 Fab' (CD20-7) was 3.8 mg/g dry cell weight, while the parent (CD20-2) was 1.3 mg/g dry cell weight. The affinity constant Ka of CD20-7 was 2.2 x 10(9) L/mol. The primary results of competitive assays by FACS showed that CD20-7 could partially block the sites through which parent antibody (HI47) bind to Raji cells. There was difference in the Raji cells (CD20+)-binding activity between the mutant CD20-7 and parent CD20-2. The site mutation of anti-CD20 Fab' gene make it possible that the anti-CD20 antibody fragment was succeeded to obtain higher expression. In this thesis, we succeeded in completing mutation and expression of anti-CD20 Fab' genes, distinguishing its biological activity, and obtaining its highly expression. These period results will lay a foundation for development of other kind of anti-CD20 engineering antibody (for instance: Fab' Diabody and miniantibody), and make it possible for anti-CD20 antibody to be applied to tumor therapy in civil in the future.