[Molecular Mechanism of Protein C Deficiency Caused by Mutations of Gene N355S, G392E, T314A].

Objective: To study the molecular mechanism of functional defect of protein C (PC) caused by point mutations of human protein C gene ( ) N355S , G392E and T314A.

Methods: The wild-type and mutant plasmids (PC, PC, PC, PC) of gene were constructed and transiently transfected into HEK293 cells. The expression of mutant proteins in vitro were tested.

[Preparation and Application of Monoclonal Antibody Against Human von Willebrand Factor Propeptide].

Objective: To develop monoclonal antibodies that can specifically recognize human von Willebrand factor (VWF) propeptide (VWFpp) in plasma, and establish a rapid and reliable method for the detection of VWFpp antigen in plasma by using the double-antibody sandwich ELISA with the obtained anti-VWFpp monoclonal antibody.

Methods: The recombinant human VWFpp (D1 and D2 regions) protein expressed in eukaryotic cells was used as immunogen to immunize BALB/c mice with routine method, so as to obtain clones of fusion cells. After screening and identification, hybridoma cell lines secreting monoclonal antibodies against VWFpp were selected, and then double-antibody sandwich ELISA assay was used to construct VWFpp antigen detection kit for the determination of VWFpp in human plasma.

[The Effect of VWF Propeptide on VWF Mutant in D1 Domain].

Objective: To investigate whether co-transfection of wild-type VWFpp with VWF mutant in D1 region is able to correct VWF defects in biosynthesis and secretion.

Methods: Four VWF mutant plasmids were single transfected into HEK 293 cells, or co-transfected into HEK 293 cells with the wild type VWFpp plasmids. The VWF in supernatant and lysate of transfected cells were analyzed by ELISA, vertical VWF multimer electrophoresis.

[Effects of the ITGA2B Nonsense Mutation (c.2659C > T, p.Q887X) on Platelet Function in a Mouse Model of Glanzmann's Thrombasthenia Generated with CRISPR/Cas9 Technology].

Objective: To construct a mouse model of Glanzmann's thrombasthenia (GT) with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation by CRISPR/Cas9 technology, and then further explore the expression and function of glycoprotein αIIbβ3 on the surface of platelet membrane.

[Effect of Recombinant Protein in the Spacer Domain on ADAMTS13 Activity].

Objective: To obtain the recombinant protein of spacer domain in von Willebrand factor cleaving protease (ADAMTS13), and further study its biological function in ADAMTS13.

Methods: The prokaryotic expression vector was constructed by using the template of plasmid with full-length ADAMTS13, and then transfected into E coli., following the induction of IPTG with the low temperature (30 ℃).

[Development of Hybridoma Cell Lines Secreting Monoclonal Antibodies against ADAMTS13].

Objective: To establish the hybridoma cell lines secreting anti-ADAMTS13-T7 monoclonal antibodies (MA6) and to construct the MAb directed against different domains of ADAMTS13-T7.

Methods: The trucated type protein ADAMTS13 of eukaryote-expressed recombinant ADAMTS13-T7 was constructed and was purified by using Ni-NTA agarose. Then the purified ADAMTS13 was used to immunize the BALB/c mice; the antiserum titer of ADAMTS13 protein of immunized mice was deteceted by ELISA.

[Identification of Monoclonal Antibodies against von Willebrand Factor Cleaving Protease (ADAMTS13) and Their Function].

Objective: To construct and identify the monoclonal antibodies against von willebrand factor cleaving protease(ADAMTS13), and to study their biological function.

Methods: BALB/c mice were immunized by purified recombinant ADAMTS13 truncated eukaryotic protein (ADAMTS13-T7). Murine anti-human ADAMTS13 monoclonal antibodies (McAbs) were constructed by standard hybridoma technology and identified by ELISA.

[Isolation, culture and characterization of outgrowth endothelial cells from the human peripheral blood].

Compared with endothelial progenitor cells, outgrowth endothelial cells (BOECs) from peripheral blood are rich in protein for blood angiogenesis and cell adhesion, similar to mature endothelial cells in biological characteristics. Moreover, they are now replacing human umbilical vein endothelial cells for the latter's limited life span and drift of phenotype, and might become a new tool for exploring the vascular abnormalities. This study was aimed to establish the protocol of producing BOECs, and then analyze the cell phenotype and function of BOECs.

[Changes of ADAMTS13 activity and vWF antigen level in patients with acute myelogenous leukemia and their significance].

This study was purposed to investigate the changes of von Willebrand factor cleaving protease (ADAMTS13) activity and vWF antigen level in patients with acute myelogenous leukemia (AML) before and after treatment and evaluate their clinical significance. Seventy-three AML patients were enrolled in this study, the sodium citrate anticoagulated plasma was collected before and after their induction chemotherapy. Fluorescence resonance energy transfer substrate vWF73 (FRETS-vWF73) assay was established to detect the plasma ADAMTS13 activity while vWF antigen level was measured by ELISA.

[Congenital afibrinogenemia caused by a novel insertion mutation in the FGB gene].

Objective: To investigate the genetic defect and its mechanism in a patient with congenital afibrinogenemia.

Methods: The plasma fibrinogen activity and antigen of the patient was determined using the Clauss method and immuno-nephelometric assay, respectively. Genomic DNA was isolated from peripheral blood of the proband and his related family members.

[Construction of ADAMTS13-pEGFP-N1 vector and its expression in HeLa cells].

This study was aimed to construct a pEGFP-N1 vector of von Willebrand factor cleaving protease (ADAMTS13, a disintegrin and metalloprotease with a thrombospondin type 1 motifs 13) so as to pave the way for further studying its synthesis and secretion. Human full-length cDNA sequence of ADAMTS13 was acquired by polymerase chain reaction (PCR) with Phusion(®) High-Fidelity (NEB), then the PCR product was double digested with EcoRI and XhoI. After digestion, the ADAMTS13 cDNA sequence was purified and recombined with the pEGFP-N1 vector.

[Increased susceptibility of recombinant type 2A von Willebrand factor mutant A1500E to proteolysis by ADAMTS13].

Objective: To investigate the susceptibility of von Willebrand factor (VWF) type 2A mutant A1500E to proteolysis by metalloprotease ADAMTS13 and to provide the direct supports for the pathogenesis of VWF mutation A1500E responsible for von Willebrand disease (VWD) type 2A.

Methods: Recombinant wild-type VWF (WT-VWF) and A1500E mutant VWF transiently expressed on transfected HeLa cell lines. Expression media were collected and concentrated, then cleaved directly by recombinant ADAMTS13 (rADAMTS13).

[Expression of vWF73 and VWF114 fragments of von Willebrand factor A2 domain and their utilization in detecting ADAMTS13 activity].

Objective: To construct the expression vectors of vWF73 and vWF114 fragments of von Willebrand factor (vWF) A2 domain, and to express glutathione S-transferase (GST) fusion proteins in E. coli, and to explore their values in measuring ADAMTS13 activity as substrates.

Methods: The DNA fragments encoding vWF73 and vWF114 were generated using PCR and separately cloned into pGEX-6P-1, a Schistosoma japonicum GST fusion expression vector.

[Prokaryotic expression and biological activity of recombinant human IL-17F/His protein].

Aim: To Prepare recombinant human IL-17F/His protein and investigate its biological activity in vitro.

Methods: The gene region of human IL-17F was cloned by RT-PCR. After identification by sequencing, the hIL-17F gene encoding function domain was cloned into expression plasmid PQE3.

[Research on the C-terminal domain of ADAMTS13 regulates its cleaving activity].

Objective: To study the influence of C-terminal domain of ADAMTS13 on its cleaving activity.

Methods: The full-length wild-type (WT) and C-terminal domain truncated type (TT, TSP8 + CUB domains were deleted) of human ADAMTS13 recombinant protein were transfected into and permanent expressed on Hela cells. Western blot and R-CBA were used to directly detect the activities of the two recombinant proteins under the static and stressed condition respectively.

Evaluation and clinical application of a new method for detecting ADAMTS13 activity.

Background: A severe deficiency of ADAMTS13 activity contributes to the pathogenesis of thrombotic thrombocytopenic purpura (TTP). Measuring the activity of ADAMTS13 is helpful for the diagnosis of TTP and the prognostic monitor in TTP patients. Most available assays are cumbersome and costly, so not easily adapted to routine laboratories.

[Inherited dysfibrinogenemia caused by Arg16His mutation in alpha chain of fibrinogen.].

Objective: To analyze the phenotype and genotype of a family with inherited dysfibrinogenemia.

Methods: Assays of coagulation, including activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT), were carried out with Stago Compact in the proband and his family members. The activity and antigen of fibrinogen in plasma were determined by Clauss and immunoturbidimetry, respectively.

[Prokaryotic expression, purification and biological activity of recombinant human IL-17/His protein].

Aim: To investigate the biological activity of recombinant human IL-17 protein in vitro.

Methods: The gene region of human IL-17 was cloned by RT-PCR. After identification by sequencing, the hIL-17 gene encoding function domain was cloned into expression plasmid PQE3.