Objective: To evaluate the effectiveness of random mutagenesis via mutator E.coli strain and error prone PCR. Methods: A ScFv containing phagemid was transformed into mutator strain XL1 Red and subjected to 7 overnight growth phases with 1/100 dilution of each phase. DNA was extracted and sequenced. The ScFv gene was also subjected to PCR mutagenesis. Mutation effects of Mg 2+ concentration, alteration of individual dNTP amounts and addition of dITP were investigated. Results: The mutation rate of 7 growth phases (about 50 cell cycles) in XL1 Red was less than 0.1%. In error prone PCR, higher concentration of Mg 2+ increased the mutation rate. Increased content of dTTP and dCTP had better effect than that of dATP and dGTP. Addition of dITP plus low concentration dATP or dGTP caused lower mutation rate. More than 2% mutation could be reached by 2 rounds PCR containing 5 mmol/L MgCl 2, 0.5 mmol/L MnCl 2, 1 mmol/L dTTP and 1 mmol/L dCTP. But the mutation showed obvious base bias with most substitutions at A/T positions and a prevalence of transition over transversion. Conclusion: Random mutagenesis in mutator strain has too low mutation rate for antibody affinity maturation. A more than 2% mutation rate can be obtained by error prone PCR, which is suitable for constructing mutated antibody libraries, but the base bias of error prone PCR should be considered.

Objective To investigate the molecular characterization of a Chinese pedigree with rare β thalassemia genotype.Methods Phenotypic analysis was performed using standard hematological tests to measure red blood cell parameters, including RBC,Hb,MCV,MCH,MCHC and RDW.SPIFE automatic Hb agarose gel electrophoresis instrument was used to measure hemoglobin fraction Hb A,Hb A2 and Hb F.The alleles of β thalassemia mutation were determined by RDB assay, and then cloning and sequencing were performed to define the mutation sites.Results The proband and his father had typical microcytic hypochromic anemia with low MCV and MCH(79.8, 63.1 fl and 19.9, 20.9 pg, respectively) and high level of Hb A2 (5.66% and 5.60%, respectively).The proband′s mother had normal MCV and MCH. β thalassemia mutation analysis with RDB assay showed that the proband had thalassemia minor resulting from double mutations on one globin gene.One showed codons 41/42 (-TTCT) mutation and the other was CAP mutation from positions +40 to +43 in the promoter region.These two mutations were inherited from his father.The genotype of the proband and his father was β41/42、CAP/βA ,and the genotype of his mother was βA/βA.Conclusions It′s rare that double mutations occur on single β globin gene, with one mutation on CD41/42(-TTCT) and the other mutation from positions +40 to +43 relative to the mRNA cap site in the promoter region.The findings enrich knowledge of the mutation spectrum of β thalassemia.

Objective:To improve the affinity of an anti TNF? scFv.Methods:Starting from an anti TNF? scFv gene a mutant phage antibody library was generated by error prone PCR.Affinity improved clones were selected and subjected to staggered extension process to shuffle the mutated sites.Mutants with further improved affinity were selected by bio panning.Affinity was judged by dot blot ELISA and thiocyanate elusion ELISA.Results:Seven affinity improved mutants were obtained from library constructed by error prone PCR.By StEP mediated shuffling of these 7 clones and via bio panning,mutants with further improved affinity were obtained.Conclusion:Combination of error prone PCR and StEP could be used to improve the affinity of antibodies. [

OBJECTIVETo elucidate the role of let-7d in regulating the biological behavior of ovarian cancer cells and their expressions of HMGA2 and ras proteins.

METHODSThe pre-let-7d sequence was synthesized and inserted into pcDNA6.2GW/EmGFPmiR and transfected into ovarian cancer IGROV1 cells to cause pre-let-7d overexpression. Real-time quantitative RT-PCR was employed to examine the expression levels of let-7d miRNA and HMGA2 mRNA, and Western blotting was performed to detect the expressions of HMGA2 and ras protein in the transfected cells. The effect of pcDNA6.2GW-let-7d transfection on IGROV1 cell proliferation was determined using MTT assay and the cell apoptosis rate was measured using flow cytometry.

RESULTSThe eukaryotic expression vector containing the target gene let-7d was successfully constructed and transfected into IGROV1 cells. The transfected cells showed a marked reduction of HMGA2 expression but a less obvious down-regulation of ras expression. Transfection with pcDNA6.2GW-let-7d to suppress the expression of HMGA2 caused alterations of the phenotype of IGROV1 cells shown by a reduced proliferative activity and increased cell apoptosis.

CONCLUSIONLet-7d plays an important role in altering the malignant cell phenotype of ovarian cancer IGROV1 cells by regulating the expression of HMGA2.

Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; HMGA2 Protein ; genetics ; metabolism ; Humans ; MicroRNAs ; genetics ; Ovarian Neoplasms ; genetics ; metabolism ; pathology ; Plasmids ; ras Proteins ; genetics ; metabolism

OX40 is a costimulatory receptor that is expressed primarily on activated CD4⁺, CD8⁺, and regulatory T cells. The ligation of OX40 to its sole ligand OX40L potentiates T cell expansion, differentiation, and activation and also promotes dendritic cells to mature to enhance their cytokine production. Therefore, the use of agonistic anti-OX40 antibodies for cancer immunotherapy has gained great interest. However, most of the agonistic anti-OX40 antibodies in the clinic are OX40L-competitive and show limited efficacy. Here, we discovered that BGB-A445, a non-ligand-competitive agonistic anti-OX40 antibody currently under clinical investigation, induced optimal T cell activation without impairing dendritic cell function. In addition, BGB-A445 dose-dependently and significantly depleted regulatory T cells in vitro and in vivo via antibody-dependent cellular cytotoxicity. In the MC38 syngeneic model established in humanized OX40 knock-in mice, BGB-A445 demonstrated robust and dose-dependent antitumor efficacy, whereas the ligand-competitive anti-OX40 antibody showed antitumor efficacy characterized by a hook effect. Furthermore, BGB-A445 demonstrated a strong combination antitumor effect with an anti-PD-1 antibody. Taken together, our findings show that BGB-A445, which does not block OX40-OX40L interaction in contrast to clinical-stage anti-OX40 antibodies, shows superior immune-stimulating effects and antitumor efficacy and thus warrants further clinical investigation.
Mice ; Animals ; Receptors, Tumor Necrosis Factor/physiology* ; Receptors, OX40 ; Membrane Glycoproteins ; Ligands ; Antibodies, Monoclonal/pharmacology* ; Antineoplastic Agents/pharmacology*